Single Bacterial Isolates Research Articles

A novel barcoded nanopore sequencing workflow of high-quality, full-length bacterial 16S amplicons for taxonomic annotation of bacterial isolates and complex microbial communities.

Due to recent improvements, Nanopore sequencing has become a promising method for experiments relying on amplicon sequencing. We describe a flexible workflow to generate and annotate high-quality, full-length 16S rDNA amplicons. We evaluated it for two applications, namely, (i) identification of bacterial isolates and (ii) species-level profiling of microbial communities. We assessed the identification of single bacterial isolates by sequencing, using a set of barcoded full-length 16S rRNA gene primer pairs (pair A), on 47 isolates encompassing multiple genera and compared those results with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based identification. Species-level community profiling was tested with two sets of barcoded full-length 16S primer pairs (A and B) and compared to the results obtained with shotgun Illumina sequencing using 27 stool samples. We developed a Nextflow pipeline to retain high-quality reads and taxonomically annotate them. We found high agreement between our workflow and MALDI-TOF data for isolate identification (positive predictive value = 0.90, Cramér's V = 0.857, and Theil's U = 0.316). For species-level community profiling, we found strong correlations (rs > 0.6) of alpha diversity indices between the two primer sets and Illumina sequencing. At the community level, we found significant but small differences when comparing sequencing techniques. Finally, we found a moderate to strong correlation when comparing the relative abundances of individual species (average rs = 0.6 and 0.533 for primers A and B). Despite identified shortcomings, the proposed workflow enabled accurate identification of single bacterial isolates and prominent features in microbial communities, making it a worthwhile alternative to MALDI-TOF MS and Illumina sequencing.IMPORTANCEA quick, robust, simple, and cost-effective method to identify bacterial isolates and communities in each sample is indispensable in the fields of microbiology and infection biology. Recent technological advances in Oxford Nanopore Technologies sequencing make this technique an attractive option considering the adaptability, portability, and cost-effectiveness of the platform, even with small sequencing batches. Here, we validated a flexible workflow to identify bacterial isolates and characterize bacterial communities using the Oxford Nanopore Technologies sequencing platform combined with the most recent v14 chemistry kits. For bacterial isolates, we compared our nanopore-based approach to matrix-assisted laser desorption ionization-time of flight mass spectrometry-based identification. For species-level profiling of complex bacterial communities, we compared our nanopore-based approach to Illumina shotgun sequencing. For reproducibility purposes, we wrapped the code used to process the sequencing data into a ready-to-use and self-contained Nextflow pipeline.

Enhanced bio-extraction of medicinally important plants using xylano-pectinolytic enzymes produced concurrently by Bacillus pumilus AJK

Medicinal plant extracts obtained by chemical methods have drawbacks such as high chemical, time and energy consumption, byproduct formation, lower quality and efficiency, high toxicity and lead to degradation of thermo-sensitive bioactive compounds. The purpose of this study was to investigate the ability of crude xylano-pectinolytic enzymes concoction in bio-extraction of medicinally important plant extracts, in order to reduce the use of chemical solvents for extraction. In this regard, various conditions including material to liquid ratio (MLR), enzyme dose, treatment time and stirring speed were optimized and several physico-chemical parameters including pH, total soluble solid (TSS), total dissolved solid (TDS), clarity, reducing sugar, polyphenolic content, viscosity and filterability were studied. The bio-extraction efficiency was found to be best at material-to-liquid ratio (MLR) 1:5-1:6 (g/mL), enzyme dose range between 15:60 to 20:80 (IU/g), treatment time range between 180- 240 min and stirring speed range between 50-60 rpm. Enzymatic extraction of medicinal plants resulted in 7-17% increase in clarity, 27-50% increase in TSS, 3-42% increase in reducing sugars, 20-42% increase in polyphenolic content, 13-53% increase in TDS, 15-22% increase in filterability along with decrease of 3-5% in pH and 18-43% in viscosity. Hence, this eco-friendly bio-extraction strategy could reduce the chemical usage, along with improvement of the physico-chemical properties of medicinally important extracts. The technique also enables the application of greener chemistry in the pharmaceuticals to optimize cleaner methods of extraction. This is the first report manifesting bio-extraction of medicinally important plant extracts using crude xylano-pectinolytic enzymatic concoction produced by a single bacterial isolate.

Retrospective analysis of antimicrobial resistance in bacterial pathogens from pet rabbits in Hong Kong, 2019-2022.

Antimicrobial resistance (AMR) is a growing threat to human and animal health. Data are limited on the prevalence of resistant bacteria in pet rabbits. Therefore, we aimed to identify prevalent bacterial infections and AMR profiles among pet rabbits in Hong Kong (HK). Our search of the CityU Veterinary Diagnostic Laboratory (VDL) database found 301 cases of pet rabbits submitted for bacteriologic and antimicrobial susceptibility testing by veterinarians at 20 exotic veterinary clinics across HK between 2019 and 2022. The rabbits were of 8 different breeds and had a median age of 6.5 y, with 54.8% males, 40.2% females, and 5% unspecified. Of the 301 samples received, 168 (55.8%) had positive bacterial growth; 125 (74.4%) had single bacterial isolates, and 43 (25.6%) had mixed cultures. Cultures included Enterococcus faecalis (21.3%) as the most frequently isolated gram-positive bacterium, followed by Streptococcus intermedius (12.5%), and Staphylococcus aureus (11.3%). The most frequently isolated gram-negative bacteria were Pseudomonas aeruginosa (18.1%), followed by Escherichia coli (8.3%), Pasteurella multocida (6.9%), and Klebsiella pneumoniae (4.2%). Approximately 83% of the isolates had acquired resistance to at least one antimicrobial agent, and 49.4% were multidrug-resistant. The isolated bacteria had high levels of resistance to penicillin (69.8%), clindamycin (47.4%), and doxycycline (46.9%). Our findings highlight the high levels of AMR in bacteria isolated from pet rabbit clinical samples in HK; many of these bacteria are zoonotic and pose a public health threat.

Screening and optimization of polyhydroxybutyrate (PHB) by Lysinibacillus fusiformis from diverse environmental sources

The need to explore microbial strains inhabiting ecosystems capable of yielding bioplastics with high production capacity and environmental sustainability is particularly of noteworthy due to the negative impacts of petrochemical plastics on humans and the environment. This study thus focused on the identification of novel polyhydroxybutyrate (PHB) producers, coupled with the screening and optimization of PHB or bioplastics for maximum yield. Bacterial strains were isolated from incinerator dump-site soil, hydrocarbon soil, rhizosphere and bulk soil samples of tomato and okra biochar plants. The identification of isolates was carried out based on biochemical and molecular methods. Screening of bacterial isolates for PHB was conducted using Sudan stain and Bromothymol techniques. Optimization analysis for maximum PHB production conditions by potential PHB strains was carried out via response surface methodology using Box-Behnken design at pH (2–11); substrates: rice bran (1–5 g); and time (24–96 h). Results showed that 112 bacterial isolates were recovered from all the samples collected. Tomato plant biochar soil recorded the highest number of bacterial isolates (37), while hydrocarbon soil yielded the lowest number of bacterial isolates (16). Predominant genera are Micrococcus, Bacillus and Staphylococcus. Lysinibacillus fusiformis was revealed to be the most potent PHB producer. Among the 112 bacteria isolated, nine isolates (5 isolates from tomato plant biochar soil, 2 bacteria from okra plant biochar soil and a single bacterial isolate from an incinerator dump site and hydrocarbon soil) exhibited positive results in both Sudan black and bromothymol tests. Box-Behnken design indicated the optimal experimental conditions for PHB synthesis to be pH 7, 72 h and substrates of rice bran of 3 g. In conclusion, Lysinibacillus fusiformis obtained from a tomato biochar plant demonstrates a high potential for bioplastic synthesis utilizing agricultural waste (rice bran) as a cost-effective as well as sustainable carbon source and could be utilized for commercial-scale PHB production.

Everything but the Kitchen Sink: An Analysis of Bacterial and Chemical Contaminants Found in Syringe Residue From People Who Inject Drugs.

People who inject drugs (PWID) are at high risk of severe wounds, invasive infections, and overdoses. To date, there are few data on the bacterial and chemical contaminants PWID are exposed to when using illicitly manufactured fentanyls and stimulants. Previously used injection drug use equipment was recovered in St Louis, Missouri, by harm reduction organizations over a 12-month period. Syringe residue was analyzed for bacterial contaminants by routine culturing followed by whole genome sequencing of single bacterial isolates. Chemical adulterants in syringe residue were identified by liquid chromatography-mass spectrometry. Bacteria were cultured from 58.75% of 160 syringes analyzed. Polymicrobial growth was common and was observed in 23.75% of samples. Bacillus cereus was the most common pathogen present and was observed in 20.6% of syringe residues, followed closely by Staphylococcus aureus at 18.8%. One hundred syringes underwent mass spectrometry, which demonstrated that chemical adulterants were common and included caffeine, diphenhydramine, lidocaine, quinine, and xylazine. Analysis of syringe residue from discarded drug use equipment demonstrates both chemical and biological contaminants, including medically important pathogens and adulterants.

The influence of the larval microbiome on susceptibility to Zika virus is mosquito genotype-dependent.

The microbiome of the mosquito Aedes aegypti is largely determined by the environment and influences mosquito susceptibility for arthropod-borne viruses (arboviruses). Larval interactions with different bacteria can have carry-over effects on adult Ae. aegypti replication of arboviruses, but little is known about the role that mosquito host genetics play in determining how larval-bacterial interactions shape Ae aegypti susceptibility to arboviruses. To address this question, we isolated single bacterial isolates and complex microbiomes from Ae. aegypti larvae from various field sites in Senegal. Either single bacterial isolates or complex microbiomes were added to two different genetic backgrounds of Ae. aegypti in a gnotobiotic larval system. Using 16S amplicon sequencing we showed that the bacterial community structure differs between the two genotypes of Ae. aegypti when given identical microbiomes, and the abundance of single bacterial taxa differed between Ae. aegypti genotypes. Using single bacterial isolates or the entire preserved complex microbiome, we tested the ability of specific larval microbiomes to drive differences in infection rates for Zika virus in different genetic backgrounds of Ae. aegypti. We observed that the proportion of Zika virus-infected adults was dependent on the interaction between the larval microbiome and Ae. aegypti host genetics. By using the larval microbiome as a component of the environment, these results demonstrate that interactions between the Ae. aegypti genotype and its environment can influence Zika virus infection. As Ae. aegypti expands and adapts to new environments under climate change, an understanding of how different genotypes interact with the same environment will be crucial for implementing arbovirus transmission control strategies.

Biodegradation of polyvinyl chloride by Citrobacter koseri isolated from superworms (Zophobas atratus larvae).

Polyvinyl chloride (PVC) is one of the widely used plastic products worldwide, and its accumulation in the natural environment has become a major global issue with regard to the environment and biotic health. There is accordingly strong demand for the development of solutions and methods for environmental remediation. Degrading plastic waste using microorganisms is an effective and eco-friendly method. However, evidence of bacteria that afford efficient biodegradation of unplasticized, pure PVC film has yet to be reported. Therefore, the biodegradation of PVC becomes very important. Here, we present results on the physicochemical and structural studies of PVC by Citrobacter koseri (C. koseri) isolated from the gut of the superworm, Zophobas atratus (Z. atratus) larvae. We also studied the biodegradability of PVC by the gut microbiota compared with C. koseri. We analyzed the microbial degradation of the PVC surface using field emission scanning electron microscopy (FE-SEM) and energy-dispersive X-ray spectroscopy (EDS) and confirmed that the physical and chemical changes were caused by C. koseri and the gut microbiota. The chemical structural changes were further investigated using X-ray photoelectron spectroscopy (XPS) and Fourier-transform-infrared (FTIR) spectroscopy, and it was confirmed that the oxidation of the PVC surface proceeded with the formation of carbonyl groups (C = O), and hydroxyl groups (-OH) by C. koseri. Additionally, the gut microbiota composed of diverse microbial species showed equal oxidation of PVC compared to C. koseri. Further, we evaluated the capabilities of single bacterial isolate and gut microbiota for pure PVC film biodegradation. Our results verified that C. koseri and the culturable microbiota from the gut of superworms present similar potential to utilize pure PVC film as a carbon source. These findings provide a potential solution for the biodegradation of unplasticized PVC.

Isolation and Identification of Gram-Positive and Gram-Negative Bacteria from Infertility Patients in Tikrit City

The objectives of the current study were conducting a statistical study to find out the number of men with primary and secondary infertility, immune infertility, and unexplained infertility in the city of Tikrit. Isolation and identification of Gram-positive and Gram-negative aerobic bacteria, and the study of some patterns of sensitivity to antibiotics of the isolated bacteria. The results showed that the percentage of primary infertility was the highest among infertility diseases, as the percentage reached 57%, while the lowest percentage was for immunological infertility, as it amounted to 7%. While the percentage of secondary and unexplained infertility was 15% and 31%, respectively. About the bacteriological study, the results showed two types of isolates, single bacterial isolates (52%), mixed bacterial isolates (38%). Percentages of isolates from E. coli and Klebsiella spp. and Staphylococcus spp. and Streptococcus spp. were 100%, 12%, 18%, and 10%, respectively. E. coli showed total (100%) resistance to amoxicillin/clavulanic acid and ampicillin. 50% resistance to Nalidixic. Otherwise, Escherichia coli showed resistance of 40%, 20%, 60%, 20% and 50% to Imipenem, Amikacin, Gentamicin, Azithromycin and Cefepime respectively. Klebsiella showed total (100%) resistance to amoxicillin/clavulanic acid and ampicillin. 40% resistance to Nalidixic. Otherwise, Klebsiella spp. showed resistance of 20%, 10%, 10%, 40% and 20% to imipenem, amikacin, gentamicin, azithromycin and cefepime respectively. Staphylococcus aureus showed complete resistance to penicillin, oxacillin and cefoxitin. 10%, 0%, 30%, 30%, 0%, 90% and 50% are resistant to gentamicin, nitrofurantoin, erythromycin, azithromycin, rifampicin, trimethoprim and ciprofloxacin respectively. Streptococcus spp. completely resistant to penicillin, oxacillin and cefoxitin. 30%, 0%, 20%, 10%, 0%, 70% and 30% are resistant to gentamicin, nitrofurantoin, erythromycin, azithromycin, rifampicin, trimethoprim and ciprofloxacin respectively.

ESBL Displace: A Protocol for an Observational Study to Identify Displacing Escherichia coli Strain Candidates from ESBL-Colonized Travel Returners Using Phenotypic, Genomic Sequencing and Metagenome Analysis

Introduction: Invading extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-PE), non-ESBL E. coli, and other bacteria form a complex environment in the gut. The duration and dynamics of ESBL-PE colonization varies among individuals. Understanding the factors associated with colonization may lead to decolonization strategies. In this study, we aim to identify (i) single E. coli strains and (ii) microbiome networks that correlate with retention or decline of colonization, and (iii) pan-sensitive E. coli strains that potentially could be used to displace ESBL-PE during colonization. Methods and analysis: We recruit healthy travellers to Southeast Asia for a one-year prospective observational follow-up study. We collect and biobank stool, serum, and peripheral blood mononuclear cells (PBMCs) at predefined timepoints. Additional information is collected with questionnaires. We determine the colonization status with ESBL-PE and non-ESBL E. coli and quantify cell densities in stools and ratios over time. We characterize multiple single bacterial isolates per patient and timepoint using whole genome sequencing (WGS) and 16S/ITS amplicon-based and shotgun metagenomics. We determine phylogenetic relationships between isolates, antimicrobial resistance (AMR; phenotypic and genotypic), and virulence genes. We describe the bacterial and fungal stool microbiome alpha and beta diversity on 16S/ITS metagenomic data. We describe patterns in microbiome dynamics to identify features associated with protection or risk of ESBL-PE colonization. Ethics and dissemination: The study is registered (clinicaltrials.gov; NCT04764500 on 09/02/2019) and approved by the Ethics Committee (EKNZ project ID 2019-00044). We will present anonymized results at conferences and in scientific journals. Bacterial sequencing data will be shared via publicly accessible databases according to FAIR principles.

Increased rate of enteric bacteria as cause of periprosthetic joint infections in patients with liver cirrhosis

IntroductionPeriprosthetic joint infections (PJI) are a major complication in joint-arthroplasty. Rifampicin is often used as an additional agent to treat PJI, because it penetrates bacterial biofilms. However, rifaximin, belonging to the same antibiotic class as rifampicin, is frequently used to prevent episodes of hepatic encephalopathy in patients with cirrhosis and may induce resistance to rifampicin. The aim of this study was to examine the microbial pattern of periprosthetic joint infections in cirrhotic patients and to test the hypothesis that intake of rifaximin increases the rate of resistance to rifampicin in periprosthetic joint infections.MethodsA cohort of cirrhotic patients and PJI (n = 25) was analysed on the characteristics of bacterial isolates from sonication and tissue analysis. In a second step a subgroup analysis on the development of rifampicin resistant bacterial specimens, depending on the intake of rifaximin (8 rifaximin intake patients vs. 13 non rifaximin intake patients) was performed.ResultsIntestinal bacteria were found in 50% of the specimens, which was significantly more frequent than in a control cohort. By comparison of the single bacterial isolates, rifampicin resistance was detected in 69.2% (9/13) of the rifaximin-intake samples. In contrast, the non-rifaximin-intake isolates only were resistant to rifampicin in 22.2% (4/18) of the cases (p = 0.01). The odds ratio for developing a rifampicin-resistance through rifaximin intake was calculated as OR = 13.5.ConclusionPeriprosthetic joint infections have a high incidence of being caused by enteric bacteria in cirrhotic patients. Due to this change in microbial pattern and the innate resistance to rifampicin of most of gram-negative bacteria, the therapy with rifampicin should be carefully considered. The association between the use of rifaximin and developed resistance to rifampicin has a major impact on the treatment of PJI.

Impact of Bacterial Phenotypic Variation with Bacteriophage therapy: A Pilot Study with Prosthetic Joint Infection Isolates

Background: Given the specificity of bacteriophage attachment receptors, a single bacterial isolate is currently utilized to match to a bacteriophage therapeutic, thereby extrapolating activity to all bacteria in vivo. Consistently, the main bacteriophage attachment receptor for Staphylococcus aureus is teichoic acid, and it is known that this receptor has phenotypic variations in different in vivo environments. Consequently, the aim of this study was to determine whether bacteriophage activity is similar across all in vivo prosthetic joint infection environments. Methods: Three patients with prosthetic joint infections who had S. aureus grow from arthrocentesis cultures and at least three deep tissue cultures were analyzed for growth inhibition with a library of 56 bacteriophages. Results: Discordant bacteriophage activity was seen across the different in vivo environments. Furthermore, bacteriophages with the most robust lytic potential to the arthrocentesis isolates usually did not demonstrate activity corresponding to all the deep tissue clinical isolates. Conclusion:Variations of bacteriophage activity can occur between the different in vivo clinical environments, which is likely secondary to different glycosylation patterns of teichoic acid. Consequently, if discordant activity is present, retreating with bacteriophages that have activity is likely needed for effective, reproducible outcomes.

Bacterial Isolates and Their Antibiotic Susceptibility Pattern among Patients with Infected Wounds Admited in Orthopaedic and Trauma Ward in Tertiary Care Hospital North-Eastern Tanzania

The hospital environment contributes to wound infections. Effects of such infections include prolonged hospitalization, increased morbidity, potential for antibiotics resistance and mortality due to sepsis. An updated knowledge of antibiotics susceptibility profiles of clinical isolates will assist both in choosing the most appropriate antibiotic treatment for wound infections and help in curbing the escalation of drug resistance. Cross sectional hospital based study, analysis of 125 pus samples collected from January 2018 to December 2020 was conducted. Identification and characterization of isolates were performed on the basis of Gram staining, microscopic characteristics, colony characteristic, and biochemical tests using standard microbiological methods. Antibiotic susceptibilities of bacterial isolates were determined by Kirby Bauer disc diffusion. A total of 125 pus samples were studied, 94 (75%) were from male patients, mean age was 38.5 (SD ± 19) years. Single bacterial isolates were recovered from 120 (96%) samples, 67 (53.6%) shows Multiple Drug Resistance (MDR) pattern, 74 (59.2%) were gram negative, the predominant organism isolated was Staphylococcus aureus 46 (36.8%). Gram negative isolates showed high resistance to ampicillin and cephalosporins. Gram positive isolates showed high resistance to erythromycin. Both gram positive and negative were found to be highly susceptible to gentamicin, amikacin, clindamycin, ciprofloxacin and amoxicillin/clavulanic acid. The study showed that Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli are the major bacteria isolated from pus samples. High proportion (53.6%) of the isolates was MDR. In the light of these findings, a change in antibiotic prescription policy is required at this hospital.

Characterization of Plant Growth Promoting Rhizobacteria (PGPR) on Capcissum annum

Plant Growth Promoting Bacteria Rhizobacteria (PGPR) is one of the potential bacteria to enhance of Capsicum annuum through inhabitation the growth of pathogenic fungi. This study aimed to characterize PGPR in chili plants (Capsicum annuum). PGPR was isolated from the soil habitat of the red chili plant in Cilegon, Indonesia. Screening was then carried out with the dual culture method on Petri dishes and tested through in vivo method on the red chili plant. The selected bacteria were characterized morphologically, biochemically, and physiologically. The results revealed that there were 14 single isolates of bacteria from the roots of the red chili plants. The five single bacterial isolates, namely Azostobacter, Azospirillum, Pseudomonas, Serratia, and Beijerinckia have good potential as PGPR based on multiple culture screening by producing clear zones and positively effect the growth of chili plants.

Rapid personalized AMR diagnostics using two-dimensional antibiotic resistance profiling strategy employing a thermometric NDM-1 biosensor

Antimicrobial resistance (AMR) threatens global public health and modern surgical medicine. Expression of β-lactamase genes is the major mechanism by which pathogens become antibiotic resistant. Pathogens expressing extended spectrum β-lactamases (ESBL) and carbapenemases (CP) are especially difficult to treat and are associated with increased hospitalization and mortality rates. Despite considerable effort, identification of ESBLs and CPs in a clinically relevant timeframe remains challenging. In this study, a two-dimensional AMR profiling assay strategy was developed employing panels of antibiotics (penicillins, cephamycins, oximino-cephalosporins and carbapenems) and β-lactamases inhibitors (avibactam and EDTA). The assay required the development of a novel biosensor that employed New Delhi metallo-β-lactamase-1 (NDM-1) as the sensing element. Functionally probing β-lactamase activity using substrates and inhibitors combinatorically increased the informational content that enabled the development of assays capable of simultaneous, differential identification of multiple β-lactamases expressed in a single bacterial isolate. More specifically, the assay enabled the simultaneous identification of ESBL and CP in mock samples, as well as in an engineered construct which co-expressed these β-lactamases. The NDM-1 biosensor assay was 16 times and 8 times more sensitive than the ESBL Nordmann/Dortet/Poirel (NDP) and Carba Nordmann/Poirel (NP) assays, respectively. In a retrospective study, NDM-1 biosensor assays were able to differentially identify ESBLs, metallo-CPs and serine-CPs β-lactamases in 23 clinical isolates with 100% accuracy. An assay algorithm was developed which accelerated data analytics reducing turnaround to <1 h. The assay strategy integrated with AI-based data analytics has the potential to provide physicians with a comprehensive readout of patient AMR status.

Effect of rhizobium isolates and nitrogen on nodulation, growth and yield in lentil

The Experiments were setup in three locations, Magura (23°3´ N 89°2´ E), Rangpur (25°7´ N 89°3´ E) and Mymensingh (24°8´ N 90°0´ E), Bangladesh to know the effect of two bacterial strains and 3 nitrogen doses on nodulation, yield and biomass production. The experimental material consists of one lentil popular variety viz., Binamasur-5 which was inoculated with two promising rhizobial starins/isolates namely LB-21 and LB-40 and three levels of nitrogen viz., 25, 50 and 75 kg N ha-1. The inoculant treatments were un-inoculated control (I0), LB-21 (I1), LB-40 (I2) and mixture of LB-21 and LB-40 (I1+I2). Results indicated that nodule number, nodule weight, straw yield and seed yield at the three locations were greater in bacterial isolates treated plants than in control plants. Results further indicated that mixture of two bacterial isolates had no superior effect against any single bacterial isolate. The two isolates (LB-21 and LB-40) had almost similar greater effect on nodule number, nodule weight, physiological parameters and seed yield with being the highest in LB-40. Application of 25 kg N ha-1 under both bacterial isolates performed the best in seed yield. Application of nitrogen at the rate of 75 kg ha-1 had adverse effect on nodulation, growth and seed yield in lentil. Therefore, for getting higher seed yield in lentil, should apply N as basal dose at the rate of 25 kg N ha-1 and the bacterial isolate, LB-40 may be registered as biofertilizer after few more trials in the different lentil growing areas of Bangladesh.

Concurrent infections of Aeromonas veronii and Vibrio cholerae in koi carp (Cyprinus carpio var. koi)

Recently, diseases of koi carp Cyprinus carpio var. koi have resulted in the mass mortality of stock at a koi carp breeding farm in Tianjin. The diseased fish exhibited obvious symptoms including skin ulceration, hemorrhages on body surface, and abdominal and intestinal dropsy. Seven Gram-negative bacterial isolates were isolated from the liver and spleen of diseased koi carp. Based on the phenotypic characteristics and sequence analysis of the 16S rRNA and gyrB genes, four isolates were identified as Aeromonas veronii and named as KCL-1 to KCL-4; three isolates were identified as Vibrio cholerae and named as KCS-1 to KCS-3. Two isolates A. veronii KCL-1 and V. cholerae KCS-1 were representatively used for the artificial experimental infection in the koi carp. Infection with single bacterial isolate or co-infection with these two isolates showed that both A. veronii KCL-1 and V. cholerae KCS-1 were virulent for the koi carp, while the former was more virulent than the latter. Meanwhile, the fish infected with the A. veronii KCL-1 and co-infected with the two isolates exhibited similar external clinical signs and histopathological changes with the naturally diseased ones. The positive result of the fluorescence in situ hybridization proved the presence of these two isolates in the spleen and liver of the co-infected fish. These results indicated that both A. veronii and V. cholerae participated in the disease outbreaks of the koi carp.

Systemic Immunological Responses Among Dental Infection Patients

Evaluating of some immunological responses among dental infection patients is essential in the clinical management of oral infection. Our study considered isolation of dental infection bacteria and quantitative evaluation of serum. IgA, IL-4, IL-7 and CD4 and CD8 molecules among dental plaque patients and control group. Oral bacteria from dental infection patients were isolated in appropriate media and diagnosed by biochemical tests and in vitro quantitative determination of serum IgA, IL-4, IL-7 and CD4 and CD8 molecules using ELISA technique. Single and mixed bacterial isolates were noted, mixed infection were (59.25%), the nature of bacteria was Gram positive cocci <i>Streptococcus viridans</i>, <i>Streptococcus pyogenes</i> and <i>Staphylococcus aureus</i>, and <i>Lactocacilli </i>spps, in addition to Gram negative rods black- pigmented bacteria, <i>Klebsilla pneumonia</i>, and <i>Esherishia coli</i>. Serum IgA was higher in patients (368.8±182.5) ng\ml than in control group (319.92±79.26) ng\ml. Serum IL-4 was higher patients (285.33±86.12) pg/ml than in control group (257.7±94.14) pg\ml. Serum IL-7 was higher in control group (19.59±4.14) pg/ml than in dental plaque patients (17.98±3.18) pg /ml. Serum CD4 molecules was higher in control group (1.371±0.5242) ng/ml than in dental plaque patients (1.326±0.1292) ng/ml. Serum CD8 molecules shows non-significant elevation in patients group 0.5825±0.02717 (ng\ml) than in control group 0.51±0.01643 (ng\ml) P≤0.05. Mean of the CD4/CD8 ratio was higher 2.783±1.126 in control group while it was 2.355±0.24 in dental plaque patients, however the differences were non-significant (P≤0.05). The present study conclude that the bacteria isolated from dental infection patients were mixed more than single infection, there were non-significant elevation in IgA, IL-4, and CD8 in patients while IL-7 and CD4 was lower in patients group than in control group, while CD4\CD8 ratio were lower in patients group, these result reflect the fact that mucosal antigen induce systemic tolerance to some extent since these bacteria present in oral cavity in early childhood. Therefor removing these bacteria always the best way to prevent such infections.

A one-year study of bacterial isolates causing Healthcare Acquired Infection (HAI) and their antibiotic resistance pattern at a private hospital in Egypt

The present study included 210 inpatients, out of them 180 were diagnosed as suspected cases of sepsis in the period over 12 months from December 2018 to December 2019, at a private hospital in Cairo, Egypt. Out of 210 investigated cases 180 (85.7%) were infected. Single bacterial isolates were recovered from 120 cases (66.7%), while 60 (33.3%) cases were found to be carrying more than one bacterial isolate. The results of the current study revealed that 34.4% (62/180) of isolates were Gram-positive and 65.6% (118/180) were Gram-negative.

Antimicrobial susceptibility of bacteria isolated from the infected wounds of patients with lymphoedema in East Wollega, Ethiopia.

BackgroundLymphoedema is caused by dysfunction of the lymphatic system resulting in accumulation of high-protein content fluid in the interstitial space. To date, the bacteria associated with wound infections of patients with lower limb lymphoedema in Ethiopia have not been studied. This study identified pathogenic bacteria involved in wound infection and assessed antimicrobial susceptibility patterns in patients with lymphoedema in Ethiopia.MethodsSwab samples were collected from the wounds of patients with lymphoedema and cultured using standard microbiological techniques. Micro-organisms were identified by colony morphology followed by identification and antimicrobial susceptibility testing using the automated VITEK 2 COMPACT Microbial Detection System.ResultsSwabs were collected from 103 patients and 84 were culture positive: 44 (52.4%) culture-positive samples showed polymicrobial growth and 40 (47.6%) grew single bacterial isolates. In total, 134 isolates were obtained, of which 26 gram-negative and 12 gram-positive bacterial species were identified. A total of 28/63 (44.4%) gram-negative isolates and 3/57 (5.3%) gram-positive isolates were multiple drug resistant. There was no resistance to ciprofloxacin, moxifloxacin or gentamycin among gram-negative or gram-positive bacteria.ConclusionIn this study, many infections were polymicrobial and showed multiple drug resistance. Fluoroquinolones and gentamycin, however, seemed to be effective against bacterial wound infection in this setting.

Reusing phosphate sludge enriched by phosphate solubilizing bacteria as biofertilizer: Growth promotion of Zea Mays

Phosphate sludge is considered as the main derivatives of the phosphate industry in Morocco, it is produced in huge quantities. Its management presents a big challenge for the phosphate industry. In this study phosphate solubilizing bacteria (PSB) were proposed as an innovative and ecological approach to solubilize phosphate sludge and promote plant growth. Four bacterial isolates ( Pseudomonas Sp. DN 13–01, Sphingobacterium suaedae T47, Bacillus pimilus X22 and Bacillus cereus 263AG5) were selected as a potent PSBs.A bacterial consortium was formed with the four selected bacteria after checking their biocompatibility. The bacterial consortium has a great phosphate solubilization ability compared to that of single bacterial isolate. The four selected bacteria were also able to produce indole acetic acid , siderophores , hydrogen cyanide , H 2 S and CO 2 . The use of 40% of the phosphate sludge increases more the growth of Zea mays than the other proportions used. All the selected bacteria were able to increase significantly the Zea mays growth whatever the proportion of the phosphate sludge used, especially when they were inoculated as a consortium which increased much more the Zea mays growth. Also, the combination of the phosphate sludge amendment at 40%and the inoculation of the bacterial consortium gave the best yield of Zea Mays growth, where the plant height, fresh areal and root biomass were raised by 406.5%, 553 and 593 respectively compared to phosphorus poor soil. The use of Moroccan phosphate sludge combined with phosphates solubilizing bacteria is an important innovative and ecological approach to promote plant growth. It needs to be further studied to re-exploit the Moroccan phosphate sludge.