Modeling Site-Specific Nucleotide Biases Affecting Himar1 Transposon Insertion Frequencies in TnSeq Data Sets

When using the Himar1 transposon to create transposon insertion mutant libraries, it is known that the transposon is restricted to insertions at TA dinucleotide sites throughout the genome, and the absence of insertions is used to infer which genes are essential (or conditionally essential) in a bacterial organism. It is widely assumed that insertions in nonessential regions are otherwise random, and this assumption is used as the basis of several methods for statistical analysis of TnSeq data.

Variations in the levels of acute-phase proteins and lactoferrin in serum and milk during bovine subclinical mastitis

Variations in the levels of acute phase proteins and lactoferrin in serum and milk for diagnosis of subclinical mastitis in dairy cows are described in this research paper. Milking animals from two organized dairy farms in Kerala, India, were screened by California Mastitis Test (CMT), Electrical Conductivity test (EC) and Somatic Cell Count (SCC) test to identify animals affected with sub clinical mastitis (SCM). The concentrations of acute phase proteins (APP) Haptoglobin (Hp), C- reactive protein (CRP), Albumin, Lactoferrin (Lf) and α- 1 acid glycoprotein (AGP) in milk and Hp, Albumin, Serum Amyloid A (SAA) and CRP in the serum of 40 normal cows and 40 cows affected with sub clinical mastitis were assessed. Solid phase ELISA was employed for assessment of all parameters except the albumin levels, for which spectrophotometry was used. The values of Hp in milk; and SAA, AGP and Lf in serum, were significantly elevated in the group with sub clinical mastitis. Such variations were found to be independent of the specific bacterial organism causing the disease. These results show that significant variations exist in the levels of acute phase proteins Hp, AGP and Lf in milk, and SAA in serum of animals affected with subclinical bovine mastitis that are not affected by specific bacterial etiology.

Isolation and Identification of Multidrug-Resistant Isolates of Uropathogenic Klebsiella spp in Dogs

The most widespread ailments in dogs are urinary tract infections (UTIs) caused by bacterial species. It is necessary to recognize the prevailing bacterial pathogens and their susceptibility to antimicrobial agents to effectively treat UTIs. The present study aimed to classify the bacterial organism that causes UTIs in dogs and their patterns of antimicrobial resistance. A total of 141 urine samples were collected from diseased dogs in Veterinary Clinical Complex LUVAS in Hisar, India. Culture, biochemical and sensitivity testing were performed for each of the urine samples based on standard method. Of the total 141 urine samples from dogs, 21 (14.9%) isolates were identified as Klebsiella spp. The isolates were found to be highly resistant to ampicillin (100%), penicillin G (100%), oxytetracycline (100%), enrofloxacin (85.7%), chloramphenicol (80.6%), ceftriaxone (76.2%) and cloxacillin (71.4%), while susceptibility was observed against gentamicin (100%), amikacin (100%) and neomycin (90.5%). In the current study, 19 out of 21 identified isolates were found to be multidrug-resistant. This study indicates that dogs in the study area are found to harbor highly resistant Klebsiella spp. Therefore, when deciding on the antibiotic regimen for UTIs cases, Vets should consider resistance profile of chosen antibacterial agents before usage in order to discourage dissemination of resistant organisms in the study area.

Frequency of Bacteriological Organism in Patients with Thoracic Empyema

Objective: To examine the frequency of bacteriological organism present in pleural fluid, in patients positive with thoracic empyema, assessed on the basis of pus culture test. Study Design: Cross-sectional study Place and Duration: Inpatient Department, Chest Medicine Ward 12, JPMC, Karachi, Pakistan for six months duration from 11 February 2019 to 10 August 2019. Methodology: One hundred and thirty nine patients diagnosed with Thoracic Empyema were included in this study. All patients included in the study shall undergo Pus Culture Test prior to identify the presence or absence of bacterial organism. Those with positive bacterial findings (mainly Staph Aureus and gram negative organisms) were further assessed for stratification with reference to their possible variable affecters. Results: There were 107 males and 32 females (%M: F ratio 77:23), with Empyema Thoracic, aged between 25-55 years and having a mean age of 36.28 year (± SD 8.206), were studied. The Pus Culture Test rate was 100% whereas duration of empyema was of average 18.38 days (± S.D 11.16). One hundred and four patients (74.82%) were carrying investigated bacteria which were staph. Aureus in 17 (12.50%) patients. GRAM NEGATIVE ORGANISMS in 87 (62.58%) patients. Echerea Coli in 12 (8.3%) patients, Pseudomonas. Aeruginosa in 46 (33.33%) patients, Klebseilla in 17(12.50%) patients, Enterococcus Species in 12 (8.3%) patients. While remaining thirty five patients (25.17%) were found to have other bacteria, including Streptococcus, Proteus Mirabillis and Acinobacter, responsible for Empyema. Conclusion: It is concluded that 74.82% patients had bacteria and among them 62.58% patients had gram negative organism. Pseudomonas Aeruginosa was the most common organism followed by Klebseilla and staph. Aureus. Keywords: Empyema, Thoracic, Pleural Effusion, Thoracentesis, Thoracostomy

Molecular Detection of Pathogens in Negative Blood Cultures in the Lao People’s Democratic Republic

ABSTRACTBloodstream infections cause substantial morbidity and mortality. However, despite clinical suspicion of such infections, blood cultures are often negative. We investigated blood cultures that were negative after 5 days of incubation for the presence of bacterial pathogens using specific (Rickettsia spp. and Leptospira spp.) and a broad-range 16S rRNA PCR. From 190 samples, 53 (27.9%) were positive for bacterial DNA. There was also a high background incidence of dengue (90/112 patient serum positive, 80.4%). Twelve samples (6.3%) were positive for Rickettsia spp., including two Rickettsia typhi. The 16S rRNA PCR gave 41 positives; Escherichia coli and Klebsiella pneumoniae were identified in 11 and eight samples, respectively, and one Leptospira species was detected. Molecular investigation of negative blood cultures can identify potential pathogens that will otherwise be missed by routine culture. Patient management would have been influenced in all 53 patients for whom a bacterial organism was identified, and 2.3–6.1% of patients would likely have had an altered final outcome. These findings warrant further study, particularly to determine the cost–benefit for routine use, ways of implementation, and timing of PCR for organisms such as Rickettsia and Leptospira, which are important pathogens in rural Asia.

Antibiotic use and presumptive pathogens in the Veterans Affairs Healthcare System

Background Empirical antibiotic use is common in the hospital. Here, we characterize patterns of antibiotic use, infectious diagnoses, and microbiological lab results among hospitalized patients and aim to quantify the proportion of antibiotic use that is potentially attributable to specific bacterial pathogens. Methods We conducted an observational study using electronic health records from acute care facilities in the United States Veterans Affairs Healthcare System. From October 2017 to September 2018, 482,381 hospitalizations for 332,657 unique patients that met all criteria were included. At least one antibiotic was administered at 202,037 (41.9%) of included hospital stays. We measured frequency of antibiotic use, microbiological specimen collection, and bacterial isolation by diagnosis category and antibiotic group. A tiered system based on specimen collection sites and diagnoses was used to attribute antibiotic use to presumptive causative organisms. Results Specimens were collected at 130,012 (64.4%) hospitalizations with any antibiotic use, and at least one bacterial organism was isolated at 35.1% of these stays. Frequency of bacterial isolation varied widely by diagnosis category and antibiotic group. Under increasingly lenient criteria, 10.2% to 31.4% of 974,733 antibiotic days-of-therapy could be linked to a potential bacterial pathogen. Conclusions Overall, the vast majority of antibiotic use could be linked to either an infectious diagnosis or microbiological specimen. Nearly half of antibiotic use occurred when there was a specimen collected but no bacterial organism identified, underscoring the need for rapid and improved diagnostics to optimize antibiotic use.

Grad-seq shines light on unrecognized RNA and protein complexes in the model bacterium Escherichia coli

Stable protein complexes, including those formed with RNA, are major building blocks of every living cell. Escherichia coli has been the leading bacterial organism with respect to global protein-protein networks. Yet, there has been no global census of RNA/protein complexes in this model species of microbiology. Here, we performed Grad-seq to establish an RNA/protein complexome, reconstructing sedimentation profiles in a glycerol gradient for ∼85% of all E. coli transcripts and ∼49% of the proteins. These include the majority of small noncoding RNAs (sRNAs) detectable in this bacterium as well as the general sRNA-binding proteins, CsrA, Hfq and ProQ. In presenting use cases for utilization of these RNA and protein maps, we show that a stable association of RyeG with 30S ribosomes gives this seemingly noncoding RNA of prophage origin away as an mRNA of a toxic small protein. Similarly, we show that the broadly conserved uncharacterized protein YggL is a 50S subunit factor in assembled 70S ribosomes. Overall, this study crucially extends our knowledge about the cellular interactome of the primary model bacterium E. coli through providing global RNA/protein complexome information and should facilitate functional discovery in this and related species.

Grad-seq shines light on unrecognized RNA and protein complexes in the model bacterium Escherichia coli

ABSTRACTStable protein complexes, including those formed with RNA, are major building blocks of every living cell. Escherichia coli has been the leading bacterial organism with respect to global protein-protein networks. Yet, there has been no global census of RNA/protein complexes in this model species of microbiology. Here, we performed Grad-seq to establish an RNA/protein complexome, reconstructing sedimentation profiles in a glycerol gradient for ~85% of all E. coli transcripts and ~49% of the proteins. These include the majority of small noncoding RNAs (sRNAs) detectable in this bacterium as well as the general sRNA-binding proteins, CsrA, Hfq and ProQ. In presenting use cases for utilization of these RNA and protein maps, we show that a stable association of RyeG with 30S ribosomes gives this seemingly noncoding RNA of prophage origin away as an mRNA of a toxic small protein. Similarly, we show that the broadly conserved uncharacterized protein YggL is a 50S subunit factor in assembled 70S ribosomes. Overall, this study crucially extends our knowledge about the cellular interactome of the primary model bacterium E. coli through providing global RNA/protein complexome information and should facilitate functional discovery in this and related species.

Severe Pulmonary Infection in a 20-Month-Old Female

Community-Acquired Pneumonia (CAP) is a common reason for hospitalization of a pediatric patient. We report a 20-month-old female admitted for suspected CAP. History included a week-long cough, fever, dyspnea, single occurrence of seizure-like activity, and a sick contact. Initial chest X-ray (CXR) showed left lower lobe pneumonia and parapneumonic effusion with a complex left pleural effusion. Ultrasound findings prompted the need for contrast-enhanced computed tomography (CT) of the chest. Contrast-enhanced CT of the chest confirmed a large pleural effusion with major atelectasis and mediastinal shift. The patient was treated with empiric antibiotics, video-assisted thoracoscopic surgical (VATS) decortication of empyema, and chest tube placement. Due to intraoperative complications, the VATS decortication was aborted and patient was transferred to the pediatric intensive care unit (PICU). A thoracentesis with culture failed to isolate a bacterial organism. Dexamethasone was started after repeat CXR showed persistent infiltrate. Subsequent contrast-enhanced CT of the chest showed a large collection of air and persistent consolidation. The patient received repeat VATS decortication and reinsertion of a chest tube. Repeat pleural fluid cultures failed to isolate a bacterial organism. Infectious disease (ID) consult recommended linezolid 140 mg Q8H for 4 weeks. Seven days after second VATS, a respiratory pathogen panel was positive for rhinovirus/enterovirus. With resolution of leukocytosis and clinical improvement, the patient was discharged with the chest tube in place and pediatric surgery outpatient follow-up. After three months, sequalae from both the infection and interventions presented .