Engineering Whole-Cell Biosensors for Enhanced Detection of Environmental Antibiotics Using a Synthetic Biology Approach.

Bacterial Two component systems have evolved with many intricate sensory apparatuses for external stimuli like light, temperature, oxygen, pH and chemical compounds. Recent studies have shown the potential of two-component regulatory systems (TCSs) of bacteria in creating synthetic regulatory circuits for several applications. Antimicrobial resistance is increasing globally in both developing and developed countries and it is one of the foremost global threats to public health. The resistance level to a broad spectrum of antibiotics is rising every year by 5-10%. In this context, TCSs controlling microbial physiology at the transcriptional level could be an appropriate candidate for monitoring the antibiotics present in the environment. This review provided a wide opportunity to gain knowledge about the TCSs available in diverse species to sense the antibiotics. Further, this review explored the EMeRALD (Engineered Modularized Receptors Activated via Ligand-induced Dimerization) based biosensors to repurpose the sensing modules from the microbial TCSs using the synthetic biology approach.

Synthesis and antitubercular activity of novel 4-arylalkyl substituted thio-, oxy- and sulfoxy-quinoline analogues targeting the cytochrome bc1 complex.

A library of 4-substituted quinolines was synthesised based on the structural features of the privileged 4-(benzylthio)-6-methoxy-2-methylquinoline scaffold. Quinoline-based chemical probes have proven to be effective anti-tuberculosis agents with the ability of inhibiting components of Mycobacterium tuberculosis (MTB) respiratory chain including the b subunit of the cytochrome bc1 complex. Novel 4-(arylalkyl)-thio, -oxy and sulfoxy-quinoline analogues were tested for their ability to inhibit the growth of MTB H37Rv and QcrB mutant strains, and the compounds mode of action was investigated. Members of the 4-subtituted thio- and sulfoxyquinoline series exhibited significant growth inhibitory activity in the high nanomolar range against wild-type MTB and induced depletion of intracellular ATP. These probes also showed reduced potency in the QcrB T313I mutant strain, thus indicating the cytochrome bc1 oxidase complex as the molecular target. Interestingly, new 4-(quinolin-2-yl)oxy-quinoline 4i was more selective for the QcrB T313I strain compared to the wild-type strain.

Optical biosensors for environmental monitoring: Recent advances and future perspectives in bacterial detection.

The environmental contamination due to bacterial proliferation vs their identification is the major deciding factor in the spread of diseases leading to pandemics. The advent of drug-resistant pathogenic contaminants in our environment has further added to the load of complications associated with their diagnosis and treatment. Obstructing the spread of such infections, prioritizes the expansion of sensor-based diagnostics, effectuating, a sturdy detection of disease-causing microbes, contaminating our surroundings in shortest possible time, with minimal expenditure. Among many sensors known, optical biosensors promote the recognition of pathogens befouling the environment through a comparatively intuitive, brisk, portable, multitudinous, and thrifty approach. This article reviews the recent progresses in optical biosensor-based systems for effective environmental monitoring. The technical and methodological perspectives of fundamental optical-sensing platforms are reviewed, combined with the pros and cons of every procedure. Eventually, the obstacles lying in the path of development of an effective optical biosensor device for bio-monitoring and its future perspectives are highlighted in the present work.

Personal Data for Public Benefit: The Regulatory Determinants of Social Licence for Technologically Enhanced Antimicrobial Resistance Surveillance.

Technologically enhanced surveillance systems have been proposed for the task of monitoring and responding to antimicrobial resistance (AMR) in both human, animal and environmental contexts. The use of these systems is in their infancy, although the advent of COVID-19 has progressed similar technologies in response to that pandemic. We conducted qualitative research to identify the Australian public's key concerns about the ethical, legal and social implications of an artificial intelligence (AI) and machine learning-enhanced One Health AMR surveillance system. Our study provides preliminary evidence of public support for AI/machine learning-enhanced One Health monitoring systems for AMR, provided that three main conditions are met: personal health care data must be deidentified; data use and access must be tightly regulated under strong governance; and the system must generate high-quality, reliable analyses to guide trusted health care decision-makers.

Using Culture Sensitivity Reports to Optimize Antimicrobial Therapy: Findings and Implications of Antimicrobial Stewardship Activity in a Hospital in Pakistan.

Background: There are concerns with inappropriate prescribing of antibiotics in hospitals especially broad spectrum in Pakistan and the subsequent impact on antimicrobial resistance rates. One recognized way to reduce inappropriate prescribing is for empiric therapy to be adjusted according to the result of culture sensitivity reports. Objective: Using culture sensitivity reports to optimize antibiotic prescribing in a teaching hospital in Pakistan. Methods: A retrospective observational study was undertaken in Ghurki Trust Teaching Hospital. A total of 465 positive cultures were taken from patients during the study period (May 2018 and December 2018). The results of pathogen identification and susceptibility testing from patient-infected sites were assessed. Additional data was collected from the patient's medical file. This included demographic data, sample type, causative microbe, antimicrobial treatment, and whether empiric or definitive treatment as well as medicine costs. Antimicrobial data was assessed using World Health Organization's Defined Daily Dose methodology. Results: A total of 497 isolates were detected from the 465 patient samples as 32 patients had polymicrobes, which included 309 g-negative rods and 188 g-positive cocci. Out of 497 isolates, the most common Gram-positive pathogen isolated was Staphylococcus aureus (Methicillin-sensitive Staphylococcus aureus) (125) (25.1%) and the most common Gram-negative pathogen was Escherichia coli (140) (28.1%). Most of the gram-negative isolates were found to be resistant to ampicillin and co-amoxiclav. Most of the Acinetobacter baumannii isolates were resistant to carbapenems. Gram-positive bacteria showed the maximum sensitivity to linezolid and vancomycin. The most widely used antibiotics for empiric therapy were cefoperazone plus sulbactam, ceftriaxone, amikacin, vancomycin, and metronidazole whereas high use of linezolid, clindamycin, meropenem, and piperacillin + tazobactam was seen in definitive treatment. Empiric therapy was adjusted in 220 (71.1%) cases of Gram-negative infections and 134 (71.2%) cases of Gram-positive infections. Compared with empiric therapy, there was a 13.8% reduction in the number of antibiotics in definitive treatment. The average cost of antibiotics in definitive treatment was less than seen with empiric treatment (8.2%) and the length of hospitalization also decreased. Conclusions: Culture sensitivity reports helped reduced antibiotic utilization and costs as well as helped select the most appropriate treatment. We also found an urgent need for implementing antimicrobial stewardship programs in hospitals and the development of hospital antibiotic guidelines to reduce unnecessary prescribing of broad-spectrum antibiotics.

Antibiotic resistance of uropathogens among the community-dwelling pregnant and nonpregnant female: a step towards antibiotic stewardship.

BACKGROUND: Indiscriminate and widespread use of antibiotics has resulted in emergence of many antibiotic-resistant organisms. Antibiotic administration during pregnancy is mostly avoided, unless there is compelling medical condition. We hypothesized that the uropathogens isolated from pregnant women would be more susceptible to antibiotics compared to those isolated from nonpregnant women, thus will be helpful in formulating separate empiric guideline for pregnant women based on the resistance pattern. METHODS: This was a prospective cross-sectional study conducted over a period of 2 years in which females with the clinical diagnosis of either cystitis or asymptomatic bacteriuria during pregnancy were included from the community settings. Uropathogen species and their antimicrobial resistance pattern were compared between the pregnant and nonpregnant groups. After accounting for centre-to-centre variation and adjusting for age and socio-economic status, the adjusted odds ratio for antibiotic resistance was calculated and compared between pregnant and nonpregnant women using logistic regression analysis. RESULTS: A total of 1758 women (pregnant: 43.3%; nonpregnant: 56.6%) were screened in the study over a period of 2 years, out of which 9.3% (163/1758) were having significant bacteriuria. Escherichia coli and Klebsiella pneumoniae were the two commonest uropathogen in both the groups; their prevalence being 83.6% in pregnant women and 85.2% in nonpregnant women, respectively. Resistance against ampicillin, cefixime, cefoxitin, ceftazidime, ceftriaxone and amoxicillin-clavulanic acid were found significantly lower in the pregnant women compared to nonpregnant. After adjusting the age and socio-economic status accounting for centre-to-centre variation, the odds of resistance for cefixime, amoxicillin-clavulanic acid and co-trimoxazole were found lower and statistically significant among the pregnant women group. CONCLUSIONS: The antimicrobial resistance was significantly higher among the community-dwelling nonpregnant women compared to pregnant women in case of few antibiotics. The study highlighted the need of building local antibiogram that could help to initiate the empirical treatment and thus prevent emergence of antimicrobial resistance.

Perillaldehyde mitigates virulence factors and biofilm formation of Pseudomonas aeruginosa clinical isolates, by acting on the quorum sensing mechanism in vitro.

AIM: The incidence of biofilm linked catheter-associated urinary tract infections is increasing worldwide and Pseudomonas aeruginosa is one of the major causes. Perillaldehyde (PLD): as a natural, widely used flavouring agent, has been reported to possess various pharmacological properties. We hypothesized that PLD can inhibit biofilm formation and virulence factor (VF) production by P. aeruginosa by hampering the quorum sensing (QS) system(s). METHODS AND RESULTS: Minimum inhibitory concentration (MIC) of PLD was assessed for standard strain and two multi-drug resistant catheter isolates of P. aeruginosa utilizing the microdilution method. Microtiter plate assay, crystal violet staining and scanning electron microscopy were used to evaluate the biofilm inhibition property. CFU was utilized to assess the antifouling property of PLD. Detection of VFs and expression analysis of virulence determinants were applied to investigate the anti-virulence activity. Gene expression and molecular docking studies were also executed to explore the QS inhibition and binding of PLD with QS receptors. In the present study, PLD has significantly inhibited biofilm formation and antivirulence activity at sub-MIC levels (2.5 and 3.5 mM) in all the tested strains. In addition, molecular docking studies revealed a significant affinity towards QS receptors. DISCUSSIONS: Perillaldehyde, being a non-toxic food flavouring agent, significantly inhibited biofilm formation and exhibited antifouling property. PLD exhibited significantly reduced levels of VFs (p < 0.001) and their respective genetic determinants (p < 0.001). Gene expression analysis and molecular docking studies confirmed the interactions of PLD to the QS receptors, indicating the plausible mechanism for the anti-virulence property. SIGNIFICANCE AND IMPACT OF STUDY: This study identified the anti-virulence potential of PLD and provided mechanistic insights. PLD can be a suitable, non-toxic candidate for countering biofilms and associated pathogens, contributing to the prevention of biofilm-associated nosocomial infections.

Increasing Helicobacter pylori clarithromycin resistance in Australia over 20 years.

BACKGROUND: Helicobacter pylori infection is responsible for considerable morbidity and mortality worldwide, and eradication rates are falling in many countries, primarily due to clarithromycin and metronidazole resistance. AIMS: There is a paucity of contemporary Australian data, which we sought to address by evaluating local rates of resistance of H. pylori to amoxicillin, clarithromycin, metronidazole and tetracycline over the past 20 years. METHODS: All gastric biopsy specimens collected at endoscopy to detect H. pylori infection at a single centre underwent routine culture and antibiotic susceptibility testing between 1998 and 2017. Specimens from 12 842 patients were cultured for H. pylori, of which 1473 positive cultures were tested for antibiotic susceptibility. RESULTS: Antibiotic resistance to clarithromycin increased by 3.7% per year (incidence rate ratio [IRR] 1.037; P = 0.014) over 20 years, with a corresponding 5.0% annual increase in minimum inhibitory concentration (MIC) (odds ratio 1.050; P < 0.001). Since 2010, average clarithromycin resistance has exceeded 20%, with >25% of isolates resistant in the past 2 years of data capture. In contrast, rates of resistance to metronidazole (35.3%), amoxicillin (0.14%) and tetracycline (0.34%) and their MIC have remained stable. Review of a representative sample (n = 120; 8%) of these patients revealed that only 5% had documented prior H. pylori eradication therapy. CONCLUSIONS: Over the past 20 years there has been a substantial rise in clarithromycin resistance, with stable metronidazole resistance and low rates of resistance to amoxicillin and tetracycline. Current first-line H. pylori eradication therapy may fail to achieve adequate eradication rates, and optimal first-line therapy in Australia should be revisited.

Characterization of a putative maltodextrin-binding protein of Streptococcus pyogenes, SPs0871 and the development of a VHH inhibitor.

Streptococcus pyogenes causes a wide range of human infections. Currently, antibiotics are the main treatment for S. pyogenes infection, but serious anti-microbial resistance requires alternative treatment options. To develop a novel strategy for treatment, we physicochemically characterized SPs0871, a putative maltose/maltodextrin-binding protein that is thought to have important roles in the pathogenesis of invasive streptococci. We obtained a variable domain of heavy chain of heavy-chain antibody, the smallest unit of an antibody, which specifically binds to SPs0871. Although the VHH completely inhibited the binding of maltodextrins to SPs0871, the inhibition did not lead to growth suppression of the bacteria. Our results provide important insights for development of VHH as an anti-streptococcal therapeutic.

Understanding the binding conformation of ceftolozane/tazobactam with Metallo-ß-lactamases VIM-5 and IMP-7 of Pseudomonas aeruginosa: A molecular docking and virtual screening process.

Pseudomonas aeruginosa (P. aeruginosa) is one of the community-acquired and healthcare-associated infections causing organisms. It has become resistant to most of the available antibiotics and is termed multi-drug resistance (MDR). There are a limited number of antibiotics are available to treat such MDR organism causing infections. The ceftolozane/tazobactam is one among the combination drug therapy (CDT) prescribed for the treatment of MDR causing infections. The resistance for the same CDT was observed in the MDR P. aeruginosa harboring VIM-5 and IMP-7 Metallo beta (ß)-lactamases (MBLs). To explore the resistance mechanism at the molecular level, docking studies were carried out for antibiotics against VIM-5 and IMP-7 MBLs. The Zn2 metal ions carry out the nucleophile attack on the carbonyl carbon of the ß-lactam ring along with conserved water molecules. To find lead compounds against the MBLs, a virtual screening process was carried out. We have employed MODELLER for structure modeling, AutoDock for molecular docking and AutoDock Vina, Molinspiration, PASS prediction & admetSAR in virtual screening. The search of low binding energy ceftolozane analogs against VIM-5 and IMP-7 MBLs has resulted in the ZINC000029060075 and ZINC000009163636 analogs. Similarly, the screening of high binding energy inhibitors against VIM-5 and IMP-7 MBLs has resulted in ZINC000003831503 and ZINC000000897247 tazobactam analogs respectively. The ADMET prediction results in the non-toxicity of the lead compounds. Our study may provide new insights for the scientist who are designing novel drugs against MDR P. aeruginosa causing infections.

WeFaceNano: a user-friendly pipeline for complete ONT sequence assembly and detection of antibiotic resistance in multi-plasmid bacterial isolates.

BACKGROUND: Bacterial plasmids often carry antibiotic resistance genes and are a significant factor in the spread of antibiotic resistance. The ability to completely assemble plasmid sequences would facilitate the localization of antibiotic resistance genes, the identification of genes that promote plasmid transmission and the accurate tracking of plasmid mobility. However, the complete assembly of plasmid sequences using the currently most widely used sequencing platform (Illumina-based sequencing) is restricted due to the generation of short sequence lengths. The long-read Oxford Nanopore Technologies (ONT) sequencing platform overcomes this limitation. Still, the assembly of plasmid sequence data remains challenging due to software incompatibility with long-reads and the error rate generated using ONT sequencing. Bioinformatics pipelines have been developed for ONT-generated sequencing but require computational skills that frequently are beyond the abilities of scientific researchers. To overcome this challenge, the authors developed 'WeFaceNano', a user-friendly Web interFace for rapid assembly and analysis of plasmid DNA sequences generated using the ONT platform. WeFaceNano includes: a read statistics report; two assemblers (Miniasm and Flye); BLAST searching; the detection of antibiotic resistance- and replicon genes and several plasmid visualizations. A user-friendly interface displays the main features of WeFaceNano and gives access to the analysis tools. RESULTS: Publicly available ONT sequence data of 21 plasmids were used to validate WeFaceNano, with plasmid assemblages and anti-microbial resistance gene detection being concordant with the published results. Interestingly, the "Flye" assembler with "meta" settings generated the most complete plasmids. CONCLUSIONS: WeFaceNano is a user-friendly open-source software pipeline suitable for accurate plasmid assembly and the detection of anti-microbial resistance genes in (clinical) samples where multiple plasmids can be present.

Genome mining of biosynthetic gene clusters intended for secondary metabolites conservation in actinobacteria.

Evolution of genome sequencing technology, on the one hand, and advancement of computational genome mining tools, on the other hand, paves way for improvement in predicting secondary metabolites. In past, numerous efforts were made concerning genome mining for recognizing secondary metabolites within the genus, but only a negligible quantity of comparative genomic reports had carried out among species of different genera. In this study, we explored potential of 24 actinobacteria species belonging to the genera, including Streptomyces, Nocardia, Micromonospora, and Saccharomonospora, to traverse diversity and distribution of Biosynthetic Gene Clusters (BGCs). Investigating results obtained from antiSMASH (Antibiotics and Secondary Metabolites Analysis Shell), NaPDoS (Natural Product Domain Seeker), and NP.searcher revealed conservation of genus-specific gene clusters among various species. E.g., NAGGN (n-acetyl glutaminyl glutamine amide) is present in Micromonospora, furan in Nocardia, melanin, and lassopeptide occur in Streptomyces. Bioactive compounds like alkyl-O-dihydro geranyl methoxy hydroquinone, SapB, desferrioxamine E, 2-Methylisoborneol, mayamycin, cyclodipeptide synthase, diisonitrile, salinichelin, hopene, ectoine and isorenieratene are highly conserved among diverse genera. Furthermore, pharmacological activity of actinobacterial derived metabolites against bacterial and fungal pathogens were illustrated. We need to accomplish large-scale analysis of natural products, including various genera of actinobacteria to deliver comprehensive intuition to overcome antibiotic resistance.

Microbes of the human eye: Microbiome, antimicrobial resistance and biofilm formation.

BACKGROUND: The review focuses on the bacteria associated with the human eye using the dual approach of detecting cultivable bacteria and the total microbiome using next generation sequencing. The purpose of this review was to highlight the connection between antimicrobial resistance and biofilm formation in ocular bacteria. METHODS: Pubmed was used as the source to catalogue culturable bacteria and ocular microbiomes associated with the normal eyes and those with ocular diseases, to ascertain the emergence of anti-microbial resistance with special reference to biofilm formation. RESULTS: This review highlights the genetic strategies used by microorganisms to evade the lethal effects of anti-microbial agents by tracing the connections between candidate genes and biofilm formation. CONCLUSION: The eye has its own microbiome which needs to be extensively studied under different physiological conditions; data on eye microbiomes of people from different ethnicities, geographical regions etc. are also needed to understand how these microbiomes affect ocular health.

Co-delivery of hesperidin and clarithromycin in a nanostructured lipid carrier for the eradication of Helicobacter pylori in vitro.

Effective and precise eradication of Helicobacter pylori (H. pylori) is the most promising approach to avoid H. pylori-related gastrointestinal disorders. The present study was conducted to demonstrate the efficacy of the co-delivery of hesperidin (Hesp) and clarithromycin (CLR) in nanostructured lipid carriers (NLCs) against H. pylori. We have produced a new delivery system by combining bioflavonoid Hesp and CLR NLCs to address the failure in single antibiotic therapies. Briefly, a blend of solid lipid, liquid lipid, and surfactant was used. Homogeneous NLCs with all the formulations showed a nano size and surface-negative charge and presented high in vitro stability and slow release of the drug even after 24 h. Bioimaging studies by scanning electron microscopy, transmission electron microscopy, and imaging flow cytometry indicated that NLCs interacted with the membrane by adhering to the outer cell membrane and disrupted the membrane that resulted in the leakage of cytoplasmic contents. The prepared NLCs provide sustained and controlled drug release that can be used to increase the rate of H. pylori eradication.

The presence of antibiotic-resistant Staphylococcus spp. and Escherichia coli in smallholder pig farms in Uganda.

BACKGROUND: The development of antimicrobial resistance is of global concern, and is commonly monitored by the analysis of certain bacteria. The aim of the present study was to study the antibiotic susceptibility in isolates of Staphylococcus spp. and Escherichia (E.) coli obtained from healthy pigs originating from nineteen herds enrolled in a study on herd health management in Lira district, northern Uganda. Skin and nasal swabs were analyzed for the presence of Staphylococcus spp., and selectively cultivated to investigate the presence of methicillin-resistant Staphylococcus (S.) aureus (MRSA), and rectal swabs were analyzed for the presence of E. coli. Antibiotic susceptibility was tested by broth micro-dilution. Information on the antibiotic usage and treatment regimens during the previous year was gathered using structured interviews and longitudinal data. RESULTS: In Staphylococcus spp., resistance to penicillin (10/19 isolates; 53%), fusidic acid (42%) and tetracycline (37%) were most commonly found. In E. coli, resistance to sulfamethoxazole (46/52 isolates; 88%), tetracycline (54%) and trimethoprim (17%) was most frequent. Methicillin-resistant S. aureus was found in one sample (1/50; 2%). Multi-drug resistant isolates of Staphylococcus spp. and E. coli were found in 54 and 47% of the herds, respectively. At the herd level, no associations could be made between antibiotic resistance and herd size or treatment regimens for either of the bacteria. CONCLUSION: In conclusion, resistance to important antibiotics frequently used in animals in Uganda was common, and the presence of MRSA was demonstrated, in Ugandan pig herds.

Prophylaxis and Treatment against Klebsiella pneumoniae: Current Insights on This Emerging Anti-Microbial Resistant Global Threat.

Klebsiella pneumoniae (Kp) is an opportunistic pathogen and the leading cause of healthcare-associated infections, mostly affecting subjects with compromised immune systems or suffering from concurrent bacterial infections. However, the dramatic increase in hypervirulent strains and the emergence of new multidrug-resistant clones resulted in Kp occurrence among previously healthy people and in increased morbidity and mortality, including neonatal sepsis and death across low- and middle-income countries. As a consequence, carbapenem-resistant and extended spectrum ß-lactamase-producing Kp have been prioritized as a critical anti-microbial resistance threat by the World Health Organization and this has renewed the interest of the scientific community in developing a vaccine as well as treatments alternative to the now ineffective antibiotics. Capsule polysaccharide is the most important virulence factor of Kp and plays major roles in the pathogenesis but its high variability (more than 100 different types have been reported) makes the identification of a universal treatment or prevention strategy very challenging. However, less variable virulence factors such as the O-Antigen, outer membrane proteins as fimbriae and siderophores might also be key players in the fight against Kp infections. Here, we review elements of the current status of the epidemiology and the molecular pathogenesis of Kp and explore specific bacterial antigens as potential targets for both prophylactic and therapeutic solutions.

Evaluation of ultra-rapid susceptibility testing of ceftolozane-tazobactam by a flow cytometry assay directly from positive blood cultures.

The urgent need for rapid antimicrobial susceptibility is broadly apparent from government reports to the lay press. Accordingly, we developed a flow-cytometry assay (FCM) for evaluating ceftolozane-tazobactam (C/T) susceptibility directly on blood cultures (BC) requiring < 2 h from flag positivity to report. The protocol was optimized with C/T-susceptible and C/T-resistant gram-negative bacilli inoculated in BC aerobic bottles (Becton-Dickinson, USA), and afterward optimized for different C/T concentrations (1/4, 2/4, 4/4, and 8/4 mg/L) for 1 h incubation (37 °C), followed by FCM and software analysis. Fluorescent membrane permeability and membrane potential dyes were comparatively used to detect early cell lesions using the CytoFLEX cytometer (Beckman-Coulter, USA). Repeatability, reproducibility, and stability of the assay up to 48 h after BC positivity were determined. Internal validation was performed in spiked BC bottles with 130 Enterobacterales and 32 Pseudomonas aeruginosa isolates from Porto University (Portugal), including 13 ATCC isolates. Additionally, 64 gram-negative bacilli recovered from positive BC at Ramon y Cajal Hospital (Madrid, Spain) were tested. Categorical agreement (CA) and analytical errors were calculated comparing FCM with broth microdilution results. Only the membrane potential dyes clearly distinguished CT-susceptible and CT-resistant isolates. Excellent repeatability, reproducibility, and inter-method concordance was observed. Overall, CA was 99.1% using EUCAST criteria with 2 major errors and 98.7% with CLSI criteria with 2 major and 1 minor errors. A new, accurate, and ultra-rapid FCM (< 2 h) for testing C/T susceptibility gave accurate results and would expand current FCM antimicrobial susceptibility assay.

Preparedness of outpatient health facilities for ambulatory treatment with all-oral short DR-TB treatment regimens in Zhytomyr, Ukraine: a cross-sectional study.

BACKGROUND: Ukraine has a high burden of drug-resistant tuberculosis (DR-TB). Mental health problems, including alcohol use disorder, are common co-morbidities. One in five DR-TB patients has human immunodeficiency virus (HIV). As part of health reform, the country is moving from inpatient care to ambulatory primary care for tuberculosis (TB). In Zhytomyr oblast, Médecins Sans Frontières (MSF) is supporting care for DR-TB patients on all-oral short DR-TB regimens. This study describes the preparedness of ambulatory care facilities in Zhytomyr oblast, Ukraine, to provide good quality ambulatory care. METHODS: This is a retrospective analysis of routinely collected programme data. Before discharge of every patient from the hospital, MSF teams assess services available at outpatient facilities using a standardised questionnaire. The assessment evaluates access, human resources, availability of medicines, infection control measures, laboratory and diagnostic services, and psychosocial support. RESULTS: We visited 68 outpatient facilities in 22 districts between June 2018 and September 2019. Twenty-seven health posts, 24 TB-units, 13 ambulatories, two family doctors and one polyclinic, serving 30% of DR-TB patients in the oblast by September 2019, were included. All facilities provided directly observed treatment, but only seven (10%) provided weekend-services. All facilities had at least one medical staff member, but TB-training was insufficient and mostly limited to TB-doctors. TB-treatment and adequate storage space were available in all facilities, but only five (8%) had ancillary medicines. HIV-positive patients had to visit a separate facility to access HIV-care. Personal protective equipment was unavailable in 32 (55%) facilities. Basic laboratory services were available in TB-units, but only four (17%) performed audiometry. Only ten (42%) TB-units had psychosocial support available, and nine (38%) offered psychiatric support. CONCLUSION: Outpatient facilities in Zhytomyr oblast are not yet prepared to provide comprehensive care for DR-TB patients. Capacity of all facilities needs strengthening with trainings, infection control measures and infrastructure. Integration of psychosocial services, treatment of co-morbidities and adverse events at the same facility are essential for successful decentralisation. The health reform is an opportunity to establish quality, patient-centred care.

Breast infections - Microbiology and treatment in an era of antibiotic resistance.

INTRODUCTION: Primary breast abscesses occur in <1% of non-lactating women, rising to 11% in women with lactational mastitis. In patients undergoing breast cancer surgery, the inflammatory response to post-operative surgical site infection (SSI) has been implicated in recurrence. Anti-microbial resistance increasingly hampers treatment in each group. AIMS: Describe the demographic and predisposing characteristics of patients with primary breast abscesses and secondary infections, identify the microbial and antimicrobial patterns and formulate an evidence-based protocol for treating breast infections. METHODS: Retrospective cohort study of all breast infections (primary and post-operative) treated at UHL from 2014 to 2017. Data collected from microbiology databases and patient records was analysed using Minitab V18. RESULTS: 537 cultures from 108 patients were analysed. 47 (43.5%) had primary abscesses, 12 (11.1%) were lactational and 49 (45.4%) were post-operative SSI. For primary infections, the mean age was 41.9 (±12.2) and reinfection rate 33%. For SSIs the mean age was 51.8 (±14.52) and reinfection rate 11.8%. Overall, 29.3% were smokers, 6.4% diabetic and 2.9% pregnant. 60 (43%) patients required radiological drainage and 2 (1%) surgical drainage. 57.5% had mixed growth. The most common isolate was Staphylococcus aureus; cultured in 16.7% of primary abscesses and 24% of SSIs. 13 empiric antibiotic regimes were prescribed before 26.4% of patients changed to 12 different targeted regimes. CONCLUSION: Breast infections are frequently polymicrobial with a wide variety of organisms isolated, suggesting the need for broad spectrum coverage until culture results become available. Based on our local culture results, the addition of clindamycin to flucloxacillin would provide excellent empiric coverage for all categories of breast infection. An evidence-based treatment guideline is required and should be formulated in close collaboration with microbiology specialists.

Emerging Anti-Microbial Resistance in Febrile Neutropenia: Is it high time to evaluate quality control measures?

OBJECTIVE: We performed a prospective analysis at our center to find out the most common organisms causing bacterial infections to establish pattern of antibiotic resistance, in order to combat febrile neutropenia effectively in the terms of outcome as well as cost. METHODS: A hospital based observational study was conducted at National Institute of Blood Diseases and bone marrow transplantation from January 2017 to December 2017. Patients presented with absolute neutrophil count (ANC) of less than 500/ml were enrolled. Data were analyzed by SPSS version 21.0. P value of <0.05 was considered statistically significant. RESULTS: In this study, a total of 242 patients from various hematological disorders were enrolled and 403 bacterial isolates were obtained. The most frequent isolated gram-negative organisms were Escherichia coli, followed by Klebsiella pneumoniae and the most prevalent gram-positive organisms were staphylococcus aureus and Enterococcus species. The antimicrobial susceptibility testing revealed that most of the Staphylococcus aureus isolates were highly resistant to methicillin (p=0.002), whereas Enterococcus species were resistant to vancomycin (p=0.000). CONCLUSION: The choice of empirical antibiotic regimen should be based on local spectrum of bacteria and their regional susceptibility pattern to improve the survival and minimize hospital stay of patients.