Characterization Of Bacterial Pathogens Research Articles

Machine Learning-Assisted Raman Spectroscopy and SERS for Bacterial Pathogen Detection: Clinical, Food Safety, and Environmental Applications

Detecting pathogenic bacteria and their phenotypes including microbial resistance is crucial for preventing infection, ensuring food safety, and promoting environmental protection. Raman spectroscopy offers rapid, seamless, and label-free identification, rendering it superior to gold-standard detection techniques such as culture-based assays and polymerase chain reactions. However, its practical adoption is hindered by issues related to weak signals, complex spectra, limited datasets, and a lack of adaptability for detection and characterization of bacterial pathogens. This review focuses on addressing these issues with recent Raman spectroscopy breakthroughs enabled by machine learning (ML), particularly deep learning methods. Given the regulatory requirements, consumer demand for safe food products, and growing awareness of risks with environmental pathogens, this study emphasizes addressing pathogen detection in clinical, food safety, and environmental settings. Here, we highlight the use of convolutional neural networks for analyzing complex clinical data and surface enhanced Raman spectroscopy for sensitizing early and rapid detection of pathogens and analyzing food safety and potential environmental risks. Deep learning methods can tackle issues with the lack of adequate Raman datasets and adaptability across diverse bacterial samples. We highlight pending issues and future research directions needed for accelerating real-world impacts of ML-enabled Raman diagnostics for rapid and accurate diagnosis and surveillance of pathogens across critical fields.

Facile syringe filter-enabled bacteria separation, enrichment, and buffer exchange for clinical isolation-free digital detection and characterization of bacterial pathogens in urine.

The development of accelerated methods for pathogen identification (ID) and antimicrobial susceptibility testing (AST) for infectious diseases is necessary to facilitate evidence-based antibiotic therapy and reduce clinical overreliance on broad-spectrum antibiotics. Towards this end, droplet-based microfluidics has unlocked remarkably rapid diagnostic assays with single-cell and single-molecule resolution. Yet, droplet platforms invariably rely on testing purified bacterial samples that have been clinically isolated after lengthy (>16 h) plating. While plating-based clinical isolation is important for enriching and separating out bacteria from background in clinical samples and also facilitating buffer exchange, it creates a diagnostic bottleneck that ultimately precludes droplet-based methods from achieving significantly accelerated times-to-result. To alleviate this bottleneck, we have developed facile syringe filter-enabled strategies for bacterial separation, enrichment, and buffer exchange from urine samples. By selecting appropriately sized filter membranes, we separated bacterial cells from background particulates in urine samples and achieved up to 91% bacterial recovery after such 1-step filtration. When interfaced with droplet-based detection of bacterial cells, 1-step filtration improved the limit of detection for bacterial ID and quantification by over an order of magnitude. We also developed a facile buffer exchange strategy to prepare bacteria in urine samples for droplet-based AST that achieved up to 10-fold bacterial enrichment during buffer exchange. Our filtration strategies, can be easily integrated into droplet workflows, enable clinical isolation-free sample-to-answer ID and AST, and significantly accelerate the turnaround of standard infectious disease diagnostic workflows.

Prevalence, the antibiogram and the frequency of virulence genes of the most predominant bacterial pathogens incriminated in calf pneumonia

The purpose of this study was to investigate the prevalence, antibiotic resistance and certain virulence genes of the most predominant bacterial pathogens causing BRD. A total of 225 calves; 55 apparently healthy and 170 diseased; were sampled. Bacteriological examination, antimicrobial susceptibility testing and PCR based detection of some virulence genes were performed. In addition, the serotyping of E. coli was performed using the slide agglutination test. The most predominant bacterial pathogens retrieved from apparently healthy calves were E. coli (16.4%) and S. aureus (10.9%), and in pneumonic calves were E. coli (23.5%), P. vulgaris (12.4%) and S. aureus (11.8%). The most prevalent virulence gene in E. coli was the fimH gene (100%), followed by eaeA gene (24.5%) and hly gene (20.4%). All the examined S. aureus strains harbored spa and coa genes; likewise, all P. multocida strains harbored toxA gene. The majority of the isolated strains displayed remarkable sensitivity to norfloxacin and enrofloxacin; furthermore, the retrieved E. coli strains exhibited multidrug-resistance to gentamicin, erythromycin, streptomycin and trimethoprim-sulphamethoxazole, in addition, the isolated S. aureus and P. aeruginosa strains showed multidrug-resistance to amoxicillin, ampicillin and tetracycline. E. coli serogroups including O18, O143, O1, and O6 were retrieved from pneumonic calves as the first report in Egypt. In conclusion, the synergism between the conventional and genotypic analysis is an effective gadget for the characterization of bacterial pathogens causing BRD. Continuous surveillance of antimicrobial susceptibility is essential to select the drug of choice due to the development of multidrug-resistant strains.

Characterization of bacterial pathogens causing fruit soft rot and stem blight of bell pepper in Georgia, USA

Bacterial soft rot is one of the major destructive diseases affecting crop production worldwide. In summer 2017, soft rot of fruit and stem blight was observed on bell pepper in commercial fields in south Georgia, USA. Bacteria were isolated from infected fruits and stems, and 5 isolates (MPS05, MPS06, PF01–2, PF04–3 and PF05–3) were characterized in the study. The isolates were identified as Pectobacterium carotovorum subsp. brasiliense (Pcb) and P. aroidearum (Pa) by morphological characteristics, biochemical and physiological tests, PCR amplification using Pcb-specific primers, and phylogenetic analysis of the 16S rDNA sequences. Pathogenicity tests on bell pepper seedlings indicated that isolates MPS05 (Pcb) and PF05–3 (Pa) were aggressive, causing plant tissue discoloration, lesions on leaves and stems, plant wilt and death. Inoculation of bell pepper fruit with MPS05 and PF05–3 at different concentrations (105, 106, 107 and 108 CFU/ml) resulted in fruit tissue maceration and complete fruit rot. To our knowledge, this study is the first report of disease caused by P. carotovorum subsp. brasiliense on pepper in the United States and the first report of P. aroidearum causing disease on pepper in the world.

Characterization of bacterial pathogens associated with milk microbiota in Egypt

Milk is a substantial source of nutrients needed by all humans across lifespan development. Given its nutritional composition, milk is considered a vehicle for various microbes including beneficial and pathogenic bacteria. In this study, 270 milk samples comprising raw cow and buffalo milk and pasteurized milk with different shelf-life durations were tested along with pasteurized organic milk for the presence of Staphylococcus aureus and Escherichia coli. Collectively, 21 E. coli and 14 S. aureus isolates were cultivated and identified from total milk samples. All E. coli and S. aureus isolates exhibited resistance to erythromycin and penicillin, respectively. Serogroups O26, O128, and O111 were the most frequently identified amongst E. coli isolates, whereas staphylococcal enterotoxins (SEs) were inconsistently produced across S. aureus isolates. The molecular profile showed clustering of 6 isolates of E. coli by harboring stx1, stx2, eaeA genes, and 5 isolates of S. aureus by mecA gene. Findings revealed the bacteriological quality of popularly consumed milk in Egypt, including raw and pasteurized milk with preference to pasteurized organic milk and 7-day shelf life (7DSL) pasteurized milk. However, raw milk and 3MSL pasteurized milk were the major sources of E. coli and S. aureus, posing a serious public health issue. Key words: Raw milk, pasteurization, Staphylococcus aureus and Escherichia coli, shelf-life.

619. High Multidrug-Resistant due to TEM and CTX-M-1 Types of Extended-Spectrum β-Lactamase and blaNDM-1 Type Carbapenemase Genes among Clinical Isolates of Gram-Negative Bacilli in Asella, Central Ethiopia

BackgroundAcute infectious diseases and sepsis are among the leading causes of mortality in Ethiopia. The lack of local data concerning causative pathogens and resistance patterns results in suboptimal empirical treatment and unfavorable clinical outcome. The objective of this study was the characterization of bacterial pathogens in hospitalized patients with febrile infections in Central Ethiopia.MethodsIn total, 684 patients ≥1 year of age with fever admitted to the Asella Teaching Hospital from April 2016 to June 2018 were included. Blood and other appropriate clinical specimens were cultured. Susceptibility testing was performed using the Kirby–Bauer method and VITEK2. Confirmation of species identification and identification of resistance genes were conducted using MALDI-ToF and PCR at a microbiology laboratory in Düsseldorf, Germany.ResultsIn total, 684 study participants were included; 54% were male and mean age was 26.7 years. Thus, the overall culture positivity rate was 7.5%. Of the 83 cultured organisms, 38(46%) were Gram-negative, 43(52%) Gram-positive, and 2(2%) Candida species. Among the 38 Gram-negative isolates, 16(42%) were E. coli, 15(39%) K. pneumoniae, and 4(11%) P. aeruginosa. Resistance against commonly used antibiotics for Gram-negative at the study site was: piperacillin/tazobactam 48%(13), ampicillin/sulbactam 93% (25), cefotaxime 89%(24), ceftazidime 74%(20), Cefipime 74%(20), meropenem 7%(2), amikacin 4% (1) and gentamicin 56%(15). Of 27 Gram-negative available for resistance-gene detection, blaNDM-1 was detected in one K. pneumoniae isolate and blaNDM-1 plus blaOXA-51 in A. baumannii. 81%(22/27) of the Gram-negative rods were confirmed to contain ESBL-genes as follows: TEM 17(77%), CTX-M-1-group 15(68%), SHV-6(27%) and CTX-M-9-group 2(9%). Among isolated S.aureus, 1(5%) was confirmed to be Methicillin-resistant S. aureus.ConclusionWe found a high prevalence (81%) of ESBL-producing bacteria and 7.4% carbapenem resistance at the study site. More than half of Gram-negative isolates had two or more mobile resistance genes. These findings warrant the need for local national multidrug-resistant surveillance. Strengthening of antimicrobial stewardship programs is needed in order to face the threat of multidrug-resistant bacteria.Disclosures All authors: No reported disclosures.

Polysaccharide-based liquid storage and transport media for non-refrigerated preservation of bacterial pathogens.

The preservation of biological samples for an extended time period of days to weeks after initial collection is important for the identification, screening, and characterization of bacterial pathogens. Traditionally, preservation relies on cold-chain infrastructure; however, in many situations this is impractical or not possible. Thus, our goal was to develop alternative bacterial sample preservation and transport media that are effective without refrigeration or external instrumentation. The viability, nucleic acid stability, and protein stability of Bacillus anthracis Sterne 34F2, Francisella novicida U112, Staphylococcus aureus ATCC 43300, and Yersinia pestis KIM D27 (pgm-) was assessed for up to 28 days. Xanthan gum (XG) prepared in PBS with L-cysteine maintained more viable F. novicida U112 cells at elevated temperature (40°C) compared to commercial reagents and buffers. Viability was maintained for all four bacteria in XG with 0.9 mM L-cysteine across a temperature range of 22–40°C. Interestingly, increasing the concentration to 9 mM L-cysteine resulted in the rapid death of S. aureus. This could be advantageous when collecting samples in the built environment where there is the potential for Staphylococcus collection and stabilization rather than other organisms of interest. F. novicida and S. aureus DNA were stable for up to 45 days upon storage at 22°C or 40°C, and direct analysis by real-time qPCR, without DNA extraction, was possible in the XG formulations. XG was not compatible with proteomic analysis via LC-MS/MS due to the high amount of residual Xanthomonas campestris proteins present in XG. Our results demonstrate that polysaccharide-based formulations, specifically XG with L-cysteine, maintain bacterial viability and nucleic acid integrity for an array of both Gram-negative and Gram-positive bacteria across ambient and elevated temperatures.

Antibiogram Analysis and Characterization of Bacterial Pathogens from Leucorrhea Patients

In recent research work bacteria were isolated from samples of leucorrhea patients admitted in Lady Wellington hospital Lahore (gynae ward) and Basheer welfare hospital Shahdara Lahore. The sampling was done from pregnant and non-pregnant leucorrhea patients aged 18 to 30 years by using sterile culture sticks from vagina. The samples were spread on agar plates and incubated for overnight, bacterial strains were isolated by streak plate method. The strains were named L1, L2, L3 and L4. Identification was carried out by various morphological and biochemical tests. Molecular characterization was also done to characterize bacteria up to species level. L1 strain was identified as Streptococcus pyogens, L2 as Staphylococcus aureus, L3 as Neisseria gonorrheae and L4 as Escherchia coli. Antibiotic resistance was analyzed by disc plate method. L1, L2 and L3 strains showed maximum sensitivity with Cefepime antibiotics having values 17.74 µg/ml, 13.63 µg/ml and 12 µg/ml respectively. L4 showed max sensitivity with Azithromycin and Cloxacillin antibiotics i.e., 6.25 µg/ml and 6.4 µg/ml respectively. Optimum pH was 6.5 for L1 and L2, while 7 for L3 and L4. Optimum temperature was 37 for all strains.

Biochemical Characterization of Bacterial Pathogens from Corneal Eye Infections and their Control via Chemotherapy, Phytotherapy and Apitherapy

Biochemical Characterization of Bacterial Pathogens from Corneal Eye Infections and their Control via Chemotherapy, Phytotherapy and Apitherapy

Advances in Clinical Sample Preparation for Identification and Characterization of Bacterial Pathogens Using Metagenomics.

Whole genome sequencing (WGS) plays an increasing role in communicable disease control through high-resolution outbreak tracing, laboratory surveillance and diagnostics. However, WGS has traditionally relied on microbial culture in order to obtain pathogen specific DNA for sequencing. This has severely limited the application of whole genome sequencing on pathogens with fastidious culturing requirements. In addition, the widespread adoption of culture-independent diagnostic tests has reduced availability of cultured isolates for confirmatory testing and surveillance. These recent developments have created demand for the implementation of techniques enabling direct sequencing of microbial genomes in clinical samples without having to culture an isolate. However, sequencing of specific organisms from clinical samples can be affected by high levels of contaminating DNA from the host and other commensal microorganisms. Several methods have been introduced for selective lysis of host cells and/or separate specific organisms from a clinical sample. This review examines the different approaches for sample preparation that have been used in diagnostic and public health laboratories for metagenomic sequencing.

Clinical detection and characterization of bacterial pathogens in the genomics era.

The availability of genome sequences obtained using next-generation sequencing (NGS) has revolutionized the field of infectious diseases. Indeed, more than 38,000 bacterial and 5,000 viral genomes have been sequenced to date, including representatives of all significant human pathogens. These tremendous amounts of data have not only enabled advances in fundamental biology, helping to understand the pathogenesis of microorganisms and their genomic evolution, but have also had implications for clinical microbiology. Here, we first review the current achievements of genomics in the development of improved diagnostic tools, including those that are now available in the clinic, such as the design of PCR assays for the detection of microbial pathogens, virulence factors or antibiotic-resistance determinants, or the design of optimized culture media for ‘unculturable’ pathogens. We then review the applications of genomics to the investigation of outbreaks, either through the design of genotyping assays or the direct sequencing of the causative strains. Finally, we discuss how genomics might change clinical microbiology in the future.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-014-0114-2) contains supplementary material, which is available to authorized users.

Characterization of bacterial pathogens in rural and urban irrigation water.

The study aimed to compare the bacteriological quality of an urban and rural irrigation water source. Bacterial counts, characterization, identification and diversity of aerobic bacteria were determined. Escherichia coli isolated from both sites was subjected to antibiotic susceptibility testing, virulence gene (Stx1/Stx2 and eae) determination and (GTG)5 Rep-PCR fingerprinting. Low mean monthly counts for aerobic spore formers, anaerobic spore formers and Staphylococcus aureus were noted although occasional spikes were observed. The most prevalent bacterial species at both sites were Bacillus spp., E. coli and Enterobacter spp. In addition, E. coli and Bacillus spp. were most prevalent in winter and summer respectively. Resistance to at least one antibiotic was 84% (rural) and 83% (urban). Highest resistance at both sites was to cephalothin and ampicillin. Prevalence of E. coli possessing at least one virulence gene (Stx1/Stx2 and eae) was 15% (rural) and 42% (urban). All (rural) and 80% (urban) of E. coli possessing virulence genes showed antibiotic resistance. Complete genetic relatedness (100%) was shown by 47% of rural and 67% of urban E. coli isolates. Results from this study show that surface irrigation water sources regardless of geographical location and surrounding land-use practices can be reservoirs of similar bacterial pathogens.

Diagnostic microarray for human and animal bacterial diseases and their virulence and antimicrobial resistance genes

Rapid diagnosis and treatment of disease is often based on the identification and characterization of causative agents derived from phenotypic characteristics. Current methods can be laborious and time-consuming, often requiring many skilled personnel and a large amount of lab space. The objective of our study was to develop a spotted microarray for rapid identification and characterization of bacterial pathogens and their antimicrobial resistance genes. Our spotted microarray consists of 489 70mer probes that detect 40 bacterial pathogens of medical, veterinary and zoonotic importance (including 15 NIAID Category A, B and C pathogens); associated genes that encode resistance for antimicrobial and metal resistance; and DNA elements that are important for horizontal gene transfer among bacteria. High specificity and reliability of the microarray was achieved for bacterial pathogens of animal and human importance by validating MDR pathogenic bacteria as pure cultures or by following their inoculation in complex and highly organic sample matrices, such as soil and manure.

Isolation Identification and Characterization of Bacterial pathogens causing Calf Diarrhea with special reference to Escherichia coli.

Isolation Identification and Characterization of Bacterial pathogens causing Calf Diarrhea with special reference to Escherichia coli.

ISOLATION AND BIOCHEMICAL CHARACTERIZATION OF BACTERIAL PATHOGEN IN COMMERCALLY IMPORTANT SHRIMP AND FISH COLLECTED FROM GHERS IN SATKHIRA DISTRICT, BANGLADESH

Investigations were carried out for isolation, enumeration and characterization of bacterial pathogens from shrimp Penaeus monodon and vetki Lates calcarifer from different ghers at Asasuni thana of Satkhira district. Shrimp and vetki were randomly sampled with a cast net from each gher individually during the period of September to December, 2005. After sampling, the live specimens of 20 shrimp and 12 fish were brought to the laboratory and immediately dissected. Muscle, hepatopancreas and gill were removed aseptically, weighed and homogenized with 0.85% NaCl solution. The homogenate was diluted using consecutive decimal dilution and inoculated on standard agar plates for enumeration of total bacterial load. A total load of 106-7 CFU g-1, 105-6 CFU g-1 and 107-10 CFU/g-1 were found in hepatopancreas, muscle and gill of shrimp and 105-6 CFU g-1, 106-9 CFU g-1and 107-10 CFU ml-1 in fish muscle, gill and slime, respectively. The total bacterial load in water of the ghers was estimated to be 107-10 CFU ml-1. A total of 32 and 20 representative isolates were selected from shrimp and fish respectively to identify them up to the genus through morphological, physiological and biochemical properties. Four genera namely, Vibrio, Micrococcus, Aeromonas and Pseudomonas were identified from the samples. Vibrio (56%) was predominantly isolated followed by Micrococcus (19%), Aeromonas (16%) and Pseudomonas (9%) in P. monodon whereas Pseudomonas (45%) dominated in L. calcarifer followed by Aeromonas (30%), Micrococcus (15%) and Vibrio (10%). High bacterial load in shrimp, fish and water of the ghers, it can be assumed that shrimp culture of Satkhira district is susceptible to bacterial contamination specially vibrio which is one of the devastating pathogens causing vibriosis in our country.

Identification and Characterization of Bacterial Pathogens Causing Bloodstream Infections by DNA Microarray

Bloodstream infections are potentially life-threatening and require rapid identification and antibiotic susceptibility testing of the causative pathogen in order to facilitate specific antimicrobial therapy. We developed a prototype DNA microarray for the identification and characterization of three important bacteremia-causing species: Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. The array consisted of 120 species-specific gene probes 200 to 800 bp in length that were amplified from recombinant plasmids. These probes represented genes encoding housekeeping proteins, virulence factors, and antibiotic resistance determinants. Evaluation with 42 clinical isolates, 3 reference strains, and 13 positive blood cultures revealed that the DNA microarray was highly specific in identifying S. aureus, E. coli, and P. aeruginosa strains and in discriminating them from closely related gram-positive and gram-negative bacterial strains also known to be etiological agents of bacteremia. We found a nearly perfect correlation between phenotypic antibiotic resistance determined by conventional susceptibility testing and genotypic antibiotic resistance by hybridization to the S. aureus resistance gene probes mecA (oxacillin-methicillin resistance), aacA-aphD (gentamicin resistance), ermA (erythromycin resistance), and blaZ (penicillin resistance) and the E. coli resistance gene probes blaTEM-106 (penicillin resistance) and aacC2 (aminoglycoside resistance). Furthermore, antibiotic resistance and virulence gene probes permitted genotypic discrimination within a species. This novel DNA microarray demonstrates the feasibility of simultaneously identifying and characterizing bacteria in blood cultures without prior amplification of target DNA or preidentification of the pathogen.

Automation of MLST Using Third-Generation Liquid-Handling Technology

The molecular characterization of bacterial pathogens of clinical significance is increasingly important. Methods, such as multilocus sequence typing (MLST), allow bacterial strains to be characterized during case clusters, for antibiotic-resistant strains to be monitored, and for the impact of new vaccines to be assessed. Our laboratory performs MLST on Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus. We have developed high-throughput automated methods to allow MLST to be performed in a time scale useful in a clinical setting. Here we describe the automation of MLST on a third-generation liquid-handling robot.

Microarray analysis of microbial virulence factors.

Hybridization with oligonucleotide microchips (microarrays) was used for discrimination among strains of Escherichia coli and other pathogenic enteric bacteria harboring various virulence factors. Oligonucleotide microchips are miniature arrays of gene-specific oligonucleotide probes immobilized on a glass surface. The combination of this technique with the amplification of genetic material by PCR is a powerful tool for the detection of and simultaneous discrimination among food-borne human pathogens. The presence of six genes (eaeA, slt-I, slt-II, fliC, rfbE, and ipaH) encoding bacterial antigenic determinants and virulence factors of bacterial strains was monitored by multiplex PCR followed by hybridization of the denatured PCR product to the gene-specific oligonucleotides on the microchip. The assay was able to detect these virulence factors in 15 Salmonella, Shigella, and E. coli strains. The results of the chip analysis were confirmed by hybridization of radiolabeled gene-specific probes to genomic DNA from bacterial colonies. In contrast, gel electrophoretic analysis of the multiplex PCR products used for the microarray analysis produced ambiguous results due to the presence of unexpected and uncharacterized bands. Our results suggest that microarray analysis of microbial virulence factors might be very useful for automated identification and characterization of bacterial pathogens.