Frontline Antibiotics Research Articles

Diagnostic performance of Typhidot RDT in diagnosis of typhoid fever and antibiotic resistance characterisation in a cross-sectional study in Southern Ghana

BackgroundTyphoid fever remains a significant public health problem contributing to significant misapplication of antibiotics in Ghana. However, there is little data on the accuracy of the commonly used serology based rapid diagnostic Typhidot test kit (Typhidot RDT) for confirming typhoid fever.MethodsWe conducted a study to assess the diagnostic accuracy of Typhidot RDT in seven clinical facilities across five regions in Southern Ghana. A total of 258 participants, clinically diagnosed with typhoid fever, were enrolled in this study. Blood and stool samples were obtained for culture, Typhidot and PCR assays. Disc diffusion antibiotic sensitivity was performed to determine the resistance pattern of Salmonella enterica isolates from positive blood and stool cultures.ResultsRecovery of S. enterica isolates was higher from stool samples (14.7%) in comparison to blood samples (1.6%). The sensitivity and specificity of Typhidot compared to blood and stool cultures was 35% (19.94%—52.65%) and 45% (38.67%—51.45%), respectively. Compared to PCR, the Typhidot had a sensitivity and a specificity of 61% and 53%, respectively. Resistance phenotyping of isolates showed broad sensitivity to the front-line antibiotics used. Resistance to ampicillin (10%), cotrimoxazole (7%), azithromycin and ciprofloxacin (< 5%) was found in some isolates.ConclusionsThese findings suggest sub-optimal performance of the Typhidot RDT for diagnosis of typhoid in Ghana with a higher chance for misdiagnosis and misapplication of antibiotics. The high proportion of isolates recovered from stool culture is consistent with the pathophysiology of bacterial shedding during the acute phase of infection, which provides a window of opportunity to control typhoid transmission.

Investigating aqueous micellar systems enhancement tools for a sensitive spectrofluorimetric determination of anti-TB: Rifampicin.

Rifampicin is a frontline antibiotic in the management of tuberculosis. Since no spectrofluorimetric methods are reported for this drug, this approach was challenged to craft a sensitive, reliable, valid, fast, and green methodology. In recent years, fluorescence spectroscopy has received a lot of interest. Its benefits include ecological greenness and analytical performance. The pharmaceutical industries greatly like this approach because of its low energy and decreased solvent usage, which make it both economical and environmentally friendly. This methodology was based on utilizing the enhanced native fluorescence of the rifampicin at 341 nm after excitation at 241 nm in a beta-cyclodextrin micellar system. Modern developments in analytical chemistry have been applied to reduce risks to the workplace and environment by using distilled water as a dilution solvent for method application and optimization. The method was found excellent green with 97 eco-scale and 0.86 AGREE scores besides an 89.6 overall whiteness score. The range of linearity for rifampicin raw material was 0.2-1.5μg·mL-1, and the average recoveries for raw material and spiked plasma were 100.15% and 99.64%, respectively. The suggested technique worked well for the commercial oral syrup of Rimactane® and did not conflict with any common additives.

Antibiotic resistance profiles of sentinel bacteria isolated from aquaculture in Cambodia

ABSTRACT The misuse of antibiotics and the emergence of antimicrobial resistance (AMR) is a concern in the aquaculture industry because it contributes to global health risks and impacts the environment. This study analyzed the AMR of sentinel bacteria associated with striped catfish (Pangasisanodon hypophthalmus) and giant snakehead (Channa micropeltes), the two main fish species reared in the pond culture in Cambodia. Phenotypic and genotypic characterization of the recovered isolates from fish, water, and sediment samples revealed the presence of bacteria, such as 22 species belonging to families Aeromonadaceae, Enterobacteriaceae, and Pseudomonadaceae. Among 48 isolates, Aeromonas caviae (n = 2), Aeromonas hydrophila (n = 2), Aeromonas ichthiosmia (n = 1), Aeromonas salmonicida (n = 4) were detected. A. salmonicida and A. hydrophilla are known as fish pathogens that occur worldwide in both fresh and marine water aquaculture. Antibiotic susceptibility testing revealed antibiotic resistance patterns of 24 (50 %) isolates among 48 isolates with higher multiple antibiotic resistance index (&amp;gt; 0.2). All the isolates of Enterobacteriaceae were susceptible to ciprofloxacin. Ciprofloxacin is a frontline antibiotic that is not recommended to use in aquaculture. Therefore, its use has to be strictly controlled. This study expands our knowledge of the AMR status in aquaculture farms which is very limited in Cambodia.

Gallein potentiates isoniazid's ability to suppress Mycobacterium tuberculosis growth.

Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis (TB), can be difficult to treat because of drug tolerance. Increased intracellular polyphosphate (polyP) in Mtb enhances tolerance to antibiotics, and capsular polyP in Neisseria gonorrhoeae potentiates resistance to antimicrobials. The mechanism by which bacteria utilize polyP to adapt to antimicrobial pressure is not known. In this study, we found that Mtb adapts to the TB frontline antibiotic isoniazid (INH) by enhancing the accumulation of cellular, extracellular, and cell surface polyP. Gallein, a broad-spectrum inhibitor of the polyphosphate kinase that synthesizes polyP, prevents this INH-induced increase in extracellular and cell surface polyP levels. Gallein and INH work synergistically to attenuate Mtb's ability to grow in in vitro culture and within human macrophages. Mtb when exposed to INH, and in the presence of INH, gallein inhibits cell envelope formation in most but not all Mtb cells. Metabolomics indicated that INH or gallein have a modest impact on levels of Mtb metabolites, but when used in combination, they significantly reduce levels of metabolites involved in cell envelope synthesis and amino acid, carbohydrate, and nucleoside metabolism, revealing a synergistic effect. These data suggest that gallein represents a promising avenue to potentiate the treatment of TB.

Characterization of the diversity of type IV secretion system-encoding plasmids in Acinetobacter

ABSTRACT The multi-drug resistant pathogen Acinetobacter baumannii has gained global attention as an important clinical challenge. Owing to its ability to survive on surfaces, its capacity for horizontal gene transfer, and its resistance to front-line antibiotics, A. baumannii has established itself as a successful pathogen. Bacterial conjugation is a central mechanism for pathogen evolution. The epidemic multidrug-resistant A. baumannii ACICU harbours a plasmid encoding a Type IV Secretion System (T4SS) with homology to the E. coli F-plasmid, and plasmids with homologous gene clusters have been identified in several A. baumannii sequence types. However the genetic and host strain diversity, global distribution, and functional ability of this group of plasmids is not fully understood. Using systematic analysis, we show that pACICU2 belongs to a group of almost 120 T4SS-encoding plasmids within four different species of Acinetobacter and one strain of Klebsiella pneumoniae from human and environmental origin, and globally distributed across 20 countries spanning 4 continents. Genetic diversity was observed both outside and within the T4SS-encoding cluster, and 47% of plasmids harboured resistance determinants, with two plasmids harbouring eleven. Conjugation studies with an extensively drug-resistant (XDR) strain showed that the XDR plasmid could be successfully transferred to a more divergent A. baumanii, and transconjugants exhibited the resistance phenotype of the plasmid. Collectively, this demonstrates that these T4SS-encoding plasmids are globally distributed and more widespread among Acinetobacter than previously thought, and that they represent an important potential reservoir for future clinical concern.

Inducing vulnerability to InhA inhibition restores isoniazid susceptibility in drug-resistant Mycobacterium tuberculosis.

Of the approximately 10 million cases of Mycobacterium tuberculosis (Mtb) infections each year, over 10% are resistant to the frontline antibiotic isoniazid (INH). INH resistance is predominantly caused by mutations that decrease the activity of the bacterial enzyme KatG, which mediates the conversion of the pro-drug INH to its active form INH-NAD. We previously discovered an inhibitor of Mtb respiration, C10, that enhances the bactericidal activity of INH, prevents the emergence of INH-resistant mutants, and re-sensitizes a collection of INH-resistant mutants to INH through an unknown mechanism. To investigate the mechanism of action of C10, we exploited the toxicity of high concentrations of C10 to select for resistant mutants. We discovered two mutations that confer resistance to the disruption of energy metabolism and allow for the growth of Mtb in high C10 concentrations, indicating that growth inhibition by C10 is associated with inhibition of respiration. Using these mutants as well as direct inhibitors of the Mtb electron transport chain, we provide evidence that inhibition of energy metabolism by C10 is neither sufficient nor necessary to potentiate killing by INH. Instead, we find that C10 acts downstream of INH-NAD synthesis, causing Mtb to become particularly sensitive to inhibition of the INH-NAD target, InhA, without changing the concentration of INH-NAD or the activity of InhA, the two predominant mechanisms of potentiating INH. Our studies revealed that there exists a vulnerability in Mtb that can be exploited to render Mtb sensitive to otherwise subinhibitory concentrations of InhA inhibitor.IMPORTANCEIsoniazid (INH) is a critical frontline antibiotic to treat Mycobacterium tuberculosis (Mtb) infections. INH efficacy is limited by its suboptimal penetration of the Mtb-containing lesion and by the prevalence of clinical INH resistance. We previously discovered a compound, C10, that enhances the bactericidal activity of INH, prevents the emergence of INH-resistant mutants, and re-sensitizes a set of INH-resistant mutants to INH. Resistance is typically mediated by katG mutations that decrease the activation of INH, which is required for INH to inhibit the essential enzyme InhA. Our current work demonstrates that C10 re-sensitizes INH-resistant katG-hypomorphs without enhancing the activation of INH. We furthermore show that C10 causes Mtb to become particularly vulnerable to InhA inhibition without compromising InhA activity on its own. Therefore, C10 represents a novel strategy to curtail the development of INH resistance and to sensitize Mtb to sub-lethal doses of INH, such as those achieved at the infection site.

Structures, Interactions and Activity of the N-Terminal Truncated Variants of Antimicrobial Peptide Thanatin.

Gram-negative bacteria are intrinsically more resistant to many frontline antibiotics, which is attributed to the permeability barrier of the outer membrane, drug efflux pumps and porins. Consequently, discovery of new small molecules antibiotics to kill drug-resistant Gram-negative bacteria presents a significant challenge. Thanatin, a 21-residue insect-derived antimicrobial peptide, is known for its potent activity against Enterobacter Gram-negative bacteria, including drug-resistant strains. Here, we investigated a 15-residue N-terminal truncated analog PM15 (P1IIYCNRRTGKCQRM15) of thanatin to determine modes of action and antibacterial activity. PM15 and the P1 to Y and A substituted variants PM15Y and PM15A delineated interactions and permeabilization of the LPS-outer membrane. In antibacterial assays, PM15 and the analogs showed growth inhibition of strains of Gram-negative bacteria that is largely dependent on the composition of the culture media. Atomic-resolution structures of PM15 and PM15Y in free solution and in complex with LPS micelle exhibited persistent β-hairpin structures similar to native thanatin. However, in complex with LPS, the structures of peptides are more compact, with extensive packing interactions among residues across the two anti-parallel strands of the β-hairpin. The docked complex of PM15/LPS revealed a parallel orientation of the peptide that may be sustained by potential ionic and van der Waals interactions with the lipid A moiety of LPS. Further, PM15 and PM15Y bind to LptAm, a monomeric functional variant of LptA, the periplasmic component of the seven-protein (A-G) complex involved in LPS transport. Taken together, the structures, target interactions and antibacterial effect of PM15 presented in the current study could be useful in designing thanatin-based peptide analogs.

Combined electrical-electrochemical phenotypic profiling of antibiotic susceptibility of in vitro biofilm models.

More than 65% of bacterial infections are caused by biofilms. However, standard biofilm susceptibility tests are not available for clinical use. All conventional biofilm models suffer from a long formation time and fail to mimic in vivo microbial biofilm conditions. Moreover, biofilms make it difficult to monitor the effectiveness of antibiotics. This work creates a powerful yet simple method to form a target biofilm and develops an innovative approach to monitoring the antibiotic's efficacy against a biofilm-associated infection. A paper-based culture platform can provide a new strategy for rapid microbial biofilm formation through capillary action. A combined electrical-electrochemical technique monitors bacterial metabolism rapidly and reliably by measuring microbial extracellular electron transfer (EET) and using electrochemical impedance spectroscopy (EIS) across a microbe-electrode interface. Three representative pathogens, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus, form their biofilms controllably within an hour. Within another hour their susceptibilities to three frontline antibiotics with different action modes (gentamicin, ciprofloxacin, and ceftazidime) are examined. Our antibiotic susceptibility testing (AST) technique provides a quantifiable minimum inhibitory concentration (MIC) of those antibiotics against the in vitro biofilm models and characterizes their action mechanisms. The results will have an important positive effect because they provide immediately actionable healthcare information at a reduced cost, revolutionizing public healthcare.

Oral β-Lactam Pairs for the Treatment of Mycobacterium avium Complex Pulmonary Disease.

Cure rates for pulmonary disease caused by the Mycobacterium avium complex (MAC) are poor. While β-lactam are front line antibiotics against Mycobacterium abscessus pulmonary disease, they have not been used or recommended to treat MAC lung infections. Through a comprehensive screen of oral β-lactams, we have discovered that selected pairs combining either a penem/carbapenem or penicillin with a cephalosporin are strongly bactericidal at clinically achieved concentrations. These dual β-lactam combinations include tebipenem and sulopenem, both in phase 3, and Food and Drug Administration-approved amoxicillin and cefuroxime. They could therefore immediately enter clinical trials or clinical practice.

Synthesis and Antibacterial Activity of Mono- and Bi-Cationic Pyridinium 1,2,4-Oxadiazoles and Triazoles.

One of the main causes of mortality in humans continues to be infectious diseases. Scientists are searching for new alternatives due to the fast increase in resistance of some harmful bacteria to the frontline antibiotics. To effectively treat pathogenic infections, it is crucial to design antibiotics that can prevent the development of pathogenic resistance. For this purpose, a set of 39 quaternary pyridinium and bis-pyridinium salts with different lengths of side alkyl or fluorinated chains, heterocyclic spacers, and counter ions were tested on diverse reference bacterial ATCC (American Type Culture Collection) strains, such as S. aureus and E. coli. Subsequently, 6 out of the 39 pyridinium salts showing relevant MIC (Minimum Inhibitory Concentration) values were tested on clinically isolated, resistant strains of S. aureus, S. epidermids, S. haemolyticus, K. pneumoniae, A. baumannii, and P. aeruginosa. Additional tests have been performed to assess if the minimum concentration detected through MIC assay may limit the growth of biofilms.

Genetic and functional enrichments associated with Enterococcus faecalis isolated from the urinary tract.

Urinary tract infection (UTI) is a global health issue that imposes a substantial burden on healthcare systems. Women are disproportionately affected by UTI, with >60% of women experiencing at least one UTI in their lifetime. UTIs can recur, particularly in postmenopausal women, leading to diminished quality of life and potentially life-threatening complications. Understanding how pathogens colonize and survive in the urinary tract is necessary to identify new therapeutic targets that are urgently needed due to rising rates of antimicrobial resistance. How Enterococcus faecalis, a bacterium commonly associated with UTI, adapts to the urinary tract remains understudied. Here, we generated a collection of high-quality closed genome assemblies of clinical urinary E. faecalis isolated from the urine of postmenopausal women that we used alongside detailed clinical metadata to perform a robust comparative genomic investigation of genetic factors that may be involved in E. faecalis survival in the urinary tract.

Multi-epitope vaccine against drug-resistant strains of Mycobacterium tuberculosis: a proteome-wide subtraction and immunoinformatics approach.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the most deadly infections in humans. The emergence of multidrug-resistant and extensively drug-resistant Mtb strains presents a global challenge. Mtb has shown resistance to many frontline antibiotics, including rifampicin, kanamycin, isoniazid, and capreomycin. The only licensed vaccine, Bacille Calmette-Guerin, does not efficiently protect against adult pulmonary tuberculosis. Therefore, it is urgently necessary to develop new vaccines to prevent infections caused by these strains. We used a subtractive proteomics approach on 23 virulent Mtb strains and identified a conserved membrane protein (MmpL4, NP_214964.1) as both a potential drug target and vaccine candidate. MmpL4 is a non-homologous essential protein in the host and is involved in the pathogen-specific pathway. Furthermore, MmpL4 shows no homology with anti-targets and has limited homology to human gut microflora, potentially reducing the likelihood of adverse effects and cross-reactivity if therapeutics specific to this protein are developed. Subsequently, we constructed a highly soluble, safe, antigenic, and stable multi-subunit vaccine from the MmpL4 protein using immunoinformatics. Molecular dynamics simulations revealed the stability of the vaccine-bound Toll-like receptor-4 complex on a nanosecond scale, and immune simulations indicated strong primary and secondary immune responses in the host. Therefore, our study identifies a new target that could expedite the design of effective therapeutics, and the designed vaccine should be validated. Future directions include an extensive molecular interaction analysis, in silico cloning, wet-lab experiments, and evaluation and comparison of the designed candidate as both a DNA vaccine and protein vaccine.

Pharmacodynamic Model of the Dynamic Response of Pseudomonas aeruginosa Biofilms to Antibacterial Treatments.

Accurate pharmacokinetic-pharmacodynamic (PK-PD) models of biofilm treatment could be used to guide formulation and administration strategies to better control bacterial lung infections. To this end, we developed a detailed pharmacodynamic model of P. aeruginosa treatment with the front-line antibiotics, tobramycin and colistin, and validated it on a detailed dataset of killing dynamics. A compartmental model structure was developed in which the key features are the diffusion of the drug through a boundary layer to the bacteria, concentration-dependent interactions with bacteria, and the passage of the bacteria through successive transit states before death. The number of transit states employed was greater for tobramycin, which is a ribosomal inhibitor, than for colistin, which disrupts bacterial membranes. For both drugs, the experimentally observed delay in the killing of bacteria following drug exposure was consistent with the sum of the diffusion time and the time for passage through the transit states. For each drug, the PD model with a single set of parameters described data across a ten-fold range of concentrations and for both continuous and transient exposure protocols, as well as for combined drug treatments. The ability to predict drug response over a range of administration protocols allows this PD model to be integrated with PK descriptions to describe in vivo antibiotic response dynamics and to predict drug delivery strategies for the improved control of bacterial lung infections.

Physicochemical considerations in the formulation development of silicone elastomer vaginal rings releasing 5-nitroimidazole drugs for the treatment of bacterial vaginosis

Bacterial vaginosis (BV) is a common dysbiosis of the human vaginal microbiota characterized by depletion of hydrogen peroxide and lactic acid-producing Lactobacillus bacteria and an overgrowth of certain facultative anaerobic bacteria. Although short-term cure rates following treatment with frontline antibiotics (most notably oral metronidazole (MNZ), clindamycin vaginal cream, and MNZ vaginal gel) are generally high, longer-term recurrence rates are an issue. The development of vaginal formulations offering continuous/sustained administration of antibiotic drugs over one or more weeks might prove useful in reducing recurrence. Here, we report the manufacture and preclinical testing of matrix-type vaginal rings offering sustained release of four 5-nitroimidazole antimicrobial drugs either being used clinically or having potential in treatment of BV – MNZ, tinidazole (TNZ), secnidazole (SNZ) and ornidazole (ONZ). All four drugs showed good compatibility with a medical-grade addition-cure silicone elastomer based upon thermal analysis experiments, and matrix-type rings containing 250 mg (3.125 %w/w) of each drug were successfully manufactured by reaction injection molding. 28-day in vitro drug release studies demonstrated root-time kinetics, with daily release rates of 25, 22, 9 and 6 mg/day½ for SNZ, ONZ, MNZ and TNZ, respectively. The rank order of drug release from rings correlated with the simple molecular permeability parameter S/V, where S is the measured drug solubility in silicone fluid and V is the drug molecular volume. The relative merits of SNZ and ONZ over MNZ (the current reference treatment) are discussed. The data support development of vaginal rings for sustained release of 5-nitroimidazole compounds for treatment of BV.

P21 Development of deep learning models for Sub-Cellular Fluctuation Imaging (SCFI): a 30 min antibiotic susceptibility test for urinary tract infections

Abstract Background We have developed a phenotypic and label-free antibiotic susceptibility test (AST) termed Sub-Cellular Fluctuation Imaging (SCFI) to address rising rates of antimicrobial resistance.1 SCFI is an advanced machine-learning enabled microscope that monitors real-time fluctuations of bacterial cell membrane in response to antibiotics. By quantifying changes in magnitude and location of light scattering caused by subcellular movement, we can detect metabolic changes that occur when bacteria are challenged with antibiotics.2–5 Here, we show that improvements to SCFI’s deep-learning models can correctly classify metabolic cell states for Escherichia coli (exponential, stationary, dead) and determine cell states for UTI-related species (E. coli, Klebsiella pneumoniae and Proteus mirabilis) with front-line antibiotics (trimethoprim and nitrofurantoin). Methods A total of 100 μL per sample is introduced to microfluidic flow chambers and immobilized using a species-specific antibody coating for 10 min. The bacterial suspension is removed, washed (to minimize non-bound cells) and incubated with 100 μL of MHB broth containing either a treated (with antibiotic) or untreated (without antibiotic) condition for 30 min. Images are captured at a laser intensity of 20 Hz for 20 s, for ≥50 individual bacterial cells per test. Convolutional neural networks (CNN) were developed to enable classifications of bacteriostatic antibiotics (trimethoprim and methicillin) on E. coli, K. pneumoniae and P. mirabilis species to determine their respective performance metrics (sensitivity, specificity, PPV and NPV). Results CNN models were developed to successfully classify treated, untreated, resistant and susceptible bacterial conditions. All conditions were tested in triplicate (n=150 cells) and demonstrated high accuracy (sensitivity 88%–98%, specificity 88%–99%, PPV 88%–99% and NPV 88%–98%) when compared with known MICs. Conclusions In these experiments we successfully demonstrated rapid (30 min) and accurate (≥90%) classification of bacterial resistance states by deep learning techniques. These data also continue to support existing literature that SCFI is an AST that is agnostic to the antibiotic class and bacterial species used. This system is undergoing product development and will be translated into a bespoke hardware system for clinical and antibiotic research applications.

Using the Galleria mellonella burn wound and infection model to identify and characterize potential wound probiotics.

Burn wound infection is the leading cause of mortality among burn wound patients. One of the most commonly isolated bacterial burn wound pathogens is Pseudomonas aeruginosa, a notorious nosocomial multidrug-resistant pathogen. As a consequence of its recalcitrance to frontline antibiotic therapy, there is an urgent need to develop alternative treatment avenues to tackle this pathogen. One potential alternative infection prevention measure is to seed the wound bed with probiotic bacteria. Several species of Lactobacillus, a common commensal bacterium, have been previously reported to display growth inhibition activity against wound pathogens. Various species of this genus have also been shown to augment the wound healing process, which makes it a promising potential therapeutic agent. Due to the complexity of the burn wound trauma and burn wound infection, an in vivo model is required for the development of novel therapeutics. There are multiple in vivo models that are currently available, the most common among them being the murine model. However, mammalian burn wound infection models are logistically challenging, do not lend themselves to screening approaches and come with significant concerns around ethics and animal welfare. Recently, an invertebrate burn wound and infection model using G. mellonella has been established. This model addresses several of the challenges of more advanced animal models, such as affordability, maintenance and reduced ethical concerns. This study validates the capacity of this model to screen for potential wound probiotics by demonstrating that a variety of Lactobacillus spp. can limit P. aeruginosa burn wound infection and improve survival.

Allele distribution and phenotypic resistance to ciprofloxacin and gentamicin among extended-spectrum β-lactamase-producing Escherichia coli isolated from the urine, stool, animals, and environments of patients with presumptive urinary tract infection in Tanzania

BackgroundAdditional antimicrobial resistance to extended-spectrum β-lactamase (ESBL)-producing E. coli exhausts treatment options. We investigated allele distribution and resistance to ciprofloxacin and gentamicin among ESBL-producing E. coli isolates from the urine, stool, animals, and environments of presumptive urinary tract infection (UTI) patients, in order to gain a crucial insight toward devising prevention and control measures and treatment guidelines.MethodsArchived ESBL-producing E. coli isolates from the urine, stool, animals, and surrounding environments of presumptive UTI patients were retrieved. Antimicrobial susceptibility profiles for ciprofloxacin and gentamicin were done followed by multiplex Polymerase chain reaction (PCR) for blaCTX-M, blaTEM, and blaSHV, to determine ESBL allele distribution. Data were analyzed using STATA version 17.ResultsA total of 472 confirmed ESBL-producing E. coli isolates from Mwanza 243 (51.5%), Kilimanjaro 143 (30.3%), and Mbeya 86 (18.2%) were analyzed. Of these, 75 (15.9%) were from urine, 199 (42.2%) from stool, 58 (12.3%) from rectal/cloaca swabs of animals, and 140 (29.7%) from surrounding environments. Out of the 472 ESBL-producing E. coli, 98.9% (467) had at least one ESBL allele. The most frequent allele was blaCTX-M, which was detected in 88.1% (416/472) of isolates, followed by the blaTEM allele, which was detected in 51.5% (243/472) of isolates. A total of 40.7% (192/472) of isolates harbored dual blaCTX-M + blaTEMalleles and only 0.2% (1/472) of isolates had dual blaCTX-M + blaSHValleles, whereas 2.3% (11/472) of isolates had a combination of all three alleles (blaCTX-M + blaTEM + blaSHV). None of the isolates harbored a combination of blaTEM + blaSHVonly. Resistance to ciprofloxacin and gentamicin was observed in 70.8% (334/472) and 46.0% (217/472) of isolates, respectively. There was a significant difference in the distribution of resistance to ciprofloxacin as well as gentamicin among ESBL-producing E. coli isolated from various sources (p-value &amp;lt; 0.001 and 0.002, respectively).ConclusionAlmost all ESBL-producing E. coli isolates carry blaCTX-M, blaTEM, and blaSHV either alone or in combination, with the most common allele being blaCTX-M.The resistance to ciprofloxacin and gentamicin, which are frontline antibiotics for UTIs among ESBL-producing E. coli, is high. This implies the need to continually revise the local guidelines used for optimal empirical therapy for UTIs, and for continual research and surveillance using one health approach.

A Rapid Method to Screen for Multi Drug Resistance (MDR) Trait in Clinical Isolates of Acinetobacter baumannii-Acyl Homoserine Lactone (AHL) Screening in CRAB and CSAB Isolates

Aims: Our proposal aimed to evaluate Acyl Homoserine Lactones (AHL) as a functional marker for Multi drug resistant (MDR) potential in clinical isolates of Acinetobacter baumannii. We investigated the AHL production potential of clinical isolates using a biosensor assay directly on a commonly used agar media.&#x0D; Place and Duration of Study: Department of Molecular Diagnostics and Biomarkers, Gleneagles Global Hospitals, Lakdikapul, Hyderabad-500004.&#x0D; Methodology: Antimicrobial drug sensitivity testing (AST) was performed on 72 clinical isolates of A. baumannii against two front-line antibiotics, Imipenem (10µg) and Meropenem (10µg), by Kirby-Bauer disk diffusion method. Production of long chain Acyl Homoserine lactone (AHLs) in the clinical isolates of A. baumannii was tested by cross streaking with the biosensor Chromobacterium violaceum mutant strain CV026 and Agrobacterium tumefaciens (NTL4pZLR4) by agar plate diffusion assay. Screening and identification of the quorum sensing mediator gene abaI was done by PCR to confirm its presence in all the 72 clinical isolates.&#x0D; Results: Out of the 72 clinical isolates, 58 were Carbapenem resistant Acinetobacter baumannii (CRAB) and 14 were Carbapenem sensitive Acinetobacter baumannii (CSAB) for AST by agar disc diffusion method. None of our isolates produced short chain AHLs whereas all the isolates could produce varying amounts of long chain AHLs. Genotypic confirmation of AHL gene was obtained by abaI gene PCR.&#x0D; Conclusion: Carbapenems are the front-line antibiotics used to treat gram negative bacterial infections in emergencies and in the critical care units of hospitals. Clinical isolates A. baumannii has innate resistance to several antibiotics due to various mechanisms, biofilms forming the first line of defense against antibiotics for the bacterium. Our study used AST to carbapenem as the leading marker for MDR, assuming the innate resistance of A. baumannii to other beta lactam antibiotics. Our study brought out certain important observations namely: a) All clinical isolates of A. baumannii produced Quorum Sensing signal molecules, the AHLs b) the clinical isolates of A. baumannii did not produce any short chain AHLs b) All the clinical isolates of A. baumannii produced long chain AHLs c) AHL production is not specific to carbapenem drug resistance because even CSAB isolates produced AHL d) AHL production is inherent to all clinical isolates of A. baumannii and it apparently indicates an underlying biofilm potential and MDR trait in these A. baumannii isolates. e) AHLs could be a universal marker for revealing MDR trait and biofilm potential in clinical microbiology AST profiling protocols.

Rat model of recalcitrant prosthetic joint infection using biofilm inocula.

Prosthetic joint infection (PJI) is a rare but devastating complication of joint arthroplasty. Biofilm formation around the prosthesis confers tolerance to antibiotics so that treatment is challenging. Most animal models of PJI use planktonic bacteria to establish the infection which fails to reproduce the pathology of chronic infection. We aimed to establish a rat model of Staphylococcus aureus PJI in male Sprague Dawley rats using biofilm inocula and demonstrate its tolerance to frontline antibiotics. Pilot studies indicated that infection could be introduced to the knee joint by a biofilm-coated pin but that handling the prosthetic without disturbing the biofilmwas difficult. We therefore developed a pin with a slotted end and used a miniature-biofilm reactor to develop mature biofilm in this niche. These biofilm-laden pins consistently produced infection of the bone and jointspace. Treatment with high dose cefazolin, 250mg/kg, starting the day of surgery reduced or cleared pin-adherent bioburden within seven days, however when escalation from 25mg/kg to 250mg/kg cefazolin treatment was delayed for 48h, rats were unable to clear the infection.To track infections, we used bioluminescentbacteria, however the bioluminescent signal did not accurately track the degree of infection in the bone and joint space as the signal did not penetrate the bone. In conclusion, we demonstrate that using a custom prosthetic pin, we can generate biofilm in a specific niche using a novel bioreactor setup and initiate a rat PJI that rapidly develops tolerance to supra-clinical doses of cefazolin. This article is protected by copyright. All rights reserved.

Hypermucoviscosity Regulator RmpD Interacts with Wzc and Controls Capsular Polysaccharide Chain Length.

Klebsiella pneumoniae is a leading cause of nosocomial infections, including pneumonia, bacteremia, and urinary tract infections. Treatment options are increasingly restricted by the high prevalence of resistance to frontline antibiotics, including carbapenems, and the recently identified plasmid-conferred colistin resistance. The classical pathotype (cKp) is responsible for most of the nosocomial infections observed globally, and these isolates are often multidrug resistant. The hypervirulent pathotype (hvKp) is a primary pathogen capable of causing community-acquired infections in immunocompetent hosts. The hypermucoviscosity (HMV) phenotype is strongly associated with the increased virulence of hvKp isolates. Recent studies demonstrated that HMV requires capsule (CPS) synthesis and the small protein RmpD but is not dependent on the increased amount of capsule associated with hvKp. Here, we identified the structure of the capsular and extracellular polysaccharide isolated from hvKp strain KPPR1S (serotype K2) with and without RmpD. We found that the polymer repeat unit structure is the same in both strains and that it is identical to the K2 capsule. However, the chain length of CPS produced by strains expressing rmpD demonstrates more uniform length. This property was reconstituted in CPS from Escherichia coli isolates that possess the same CPS biosynthesis pathway as K. pneumoniae but naturally lack rmpD. Furthermore, we demonstrate that RmpD binds Wzc, a conserved capsule biosynthesis protein required for CPS polymerization and export. Based on these observations, we present a model for how the interaction of RmpD with Wzc could impact CPS chain length and HMV. IMPORTANCE Infections caused by Klebsiella pneumoniae continue to be a global public health threat; the treatment of these infections is complicated by the high frequency of multidrug resistance. K. pneumoniae produces a polysaccharide capsule required for virulence. Hypervirulent isolates also have a hypermucoviscous (HMV) phenotype that increases virulence, and we recently demonstrated that a horizontally acquired gene, rmpD, is required for HMV and hypervirulence but that the identity of the polymeric product(s) in HMV isolates is uncertain. Here, we demonstrate that RmpD regulates capsule chain length and interacts with Wzc, a part of the capsule polymerization and export machinery shared by many pathogens. We further show that RmpD confers HMV and regulates capsule chain length in a heterologous host (E. coli). As Wzc is a conserved protein found in many pathogens, it is possible that RmpD-mediated HMV and increased virulence may not be restricted to K. pneumoniae.