Coronavirus Disease 2019 and Airborne Transmission: Science Rejected, Lives Lost. Can Society Do Better?

This is an account that should be heard of an important struggle: the struggle of a large group of experts who came together at the beginning of the COVID-19 pandemic to warn the world about the risk of airborne transmission and the consequences of ignoring it. We alerted the World Health Organization about the potential significance of the airborne transmission of SARS-CoV-2 and the urgent need to control it, but our concerns were dismissed. Here we describe how this happened and the consequences. We hope that by reporting this story we can raise awareness of the importance of interdisciplinary collaboration and the need to be open to new evidence, and to prevent it from happening again. Acknowledgement of an issue, and the emergence of new evidence related to it, is the first necessary step towards finding effective mitigation solutions.

Airborne transmission of respiratory viruses including severe acute respiratory syndrome coronavirus 2.

PURPOSE OF REVIEW: The coronavirus disease 2019 pandemic has had a wide-ranging and profound impact on how we think about the transmission of respiratory viruses This review outlines the basis on which we should consider all respiratory viruses as aerosol-transmissible infections, in order to improve our control of these pathogens in both healthcare and community settings. RECENT FINDINGS: We present recent studies to support the aerosol transmission of severe acute respiratory syndrome coronavirus 2, and some older studies to demonstrate the aerosol transmissibility of other, more familiar seasonal respiratory viruses. SUMMARY: Current knowledge on how these respiratory viruses are transmitted, and the way we control their spread, is changing. We need to embrace these changes to improve the care of patients in hospitals and care homes including others who are vulnerable to severe disease in community settings.

Modeling fomite-mediated SARS-CoV-2 exposure through personal protective equipment doffing in a hospital environment.

Self-contamination during doffing of personal protective equipment (PPE) is a concern for healthcare workers (HCW) following SARS-CoV-2-positive patient care. Staff may subconsciously become contaminated through improper glove removal; so, quantifying this exposure is critical for safe working procedures. HCW surface contact sequences on a respiratory ward were modeled using a discrete-time Markov chain for: IV-drip care, blood pressure monitoring, and doctors' rounds. Accretion of viral RNA on gloves during care was modeled using a stochastic recurrence relation. In the simulation, the HCW then doffed PPE and contaminated themselves in a fraction of cases based on increasing caseload. A parametric study was conducted to analyze the effect of: (1a) increasing patient numbers on the ward, (1b) the proportion of COVID-19 cases, (2) the length of a shift, and (3) the probability of touching contaminated PPE. The driving factors for the exposure were surface contamination and the number of surface contacts. The results simulate generally low viral exposures in most of the scenarios considered including on 100% COVID-19 positive wards, although this is where the highest self-inoculated dose is likely to occur with median 0.0305 viruses (95% CI =0-0.6 viruses). Dose correlates highly with surface contamination showing that this can be a determining factor for the exposure. The infection risk resulting from the exposure is challenging to estimate, as it will be influenced by the factors such as virus variant and vaccination rates.

What were the historical reasons for the resistance to recognizing airborne transmission during the COVID-19 pandemic?

The question of whether SARS-CoV-2 is mainly transmitted by droplets or aerosols has been highly controversial. We sought to explain this controversy through a historical analysis of transmission research in other diseases. For most of human history, the dominant paradigm was that many diseases were carried by the air, often over long distances and in a phantasmagorical way. This miasmatic paradigm was challenged in the mid to late 19th century with the rise of germ theory, and as diseases such as cholera, puerperal fever, and malaria were found to actually transmit in other ways. Motivated by his views on the importance of contact/droplet infection, and the resistance he encountered from the remaining influence of miasma theory, prominent public health official Charles Chapin in 1910 helped initiate a successful paradigm shift, deeming airborne transmission most unlikely. This new paradigm became dominant. However, the lack of understanding of aerosols led to systematic errors in the interpretation of research evidence on transmission pathways. For the next five decades, airborne transmission was considered of negligible or minor importance for all major respiratory diseases, until a demonstration of airborne transmission of tuberculosis (which had been mistakenly thought to be transmitted by droplets) in 1962. The contact/droplet paradigm remained dominant, and only a few diseases were widely accepted as airborne before COVID-19: those that were clearly transmitted to people not in the same room. The acceleration of interdisciplinary research inspired by the COVID-19 pandemic has shown that airborne transmission is a major mode of transmission for this disease, and is likely to be significant for many respiratory infectious diseases.

Staphylococcus aureus nasal colonization among dental health care workers in Northern Germany (StaphDent study).

Methicillin-resistant Staphylococcus aureus (MRSA) can colonize dental patients and students, however, studies on the prevalence of MRSA and methicillin-susceptible S. aureus (MSSA) among dental health care workers (DHCW) including use of personal protective equipment (PPE) are scarce. We conducted an observational study (StaphDent study) to (I) determine the prevalence of MRSA and MSSA colonization in DHCW in the region of Mecklenburg Western-Pomerania, Germany, (II) resolve the S. aureus population structure to gain hints on possible transmission events between co-workers, and (III) clarify use of PPE. Nasal swabs were obtained from dentists (n = 149), dental assistants (n = 297) and other dental practice staff (n = 38). Clonal relatedness of MSSA isolates was investigated using spa typing and, in some cases, whole genome sequencing (WGS). PPE use was assessed by questionnaire. While 22.3% (108/485) of the participants were colonized with MSSA, MRSA was not detected. MSSA prevalence was not associated with size of dental practices, gender, age, or duration of employment. The identified 61 spa types grouped into 17 clonal complexes and four sequence types. Most spa types (n = 47) were identified only once. In ten dental practices one spa type occurred twice. WGS data analysis confirmed a close clonal relationship for 4/10 isolate pairs. PPE was regularly used by most dentists and assistants. To conclude, the failure to recover MRSA from DHCW reflects the low MRSA prevalence in this region. Widespread PPE use suggests adherence to routine hygiene protocols. Compared to other regional HCW MRSA rates the consequent usage of PPE seems to be protective.

What is the relationship between indoor air quality parameters and airborne microorganisms in hospital environments? A systematic review and meta-analysis.

Airborne microorganisms in hospitals have been associated with several hospital-acquired infections (HAIs), and various measures of indoor air quality (IAQ) parameters such as temperature, relative humidity, carbon dioxide (CO2 ), particle mass concentration, and particle size have been linked to pathogen survival or mitigation of pathogen spread. To investigate whether there are quantitative relationships between the concentration of airborne microorganisms and the IAQ in the hospital environment. Web of Science, Scopus and PubMed databases were searched for studies reporting airborne microbial levels and any IAQ parameter(s) in hospital environments, from database inception to October 2020. Pooled effect estimates were determined via random-effects models. Seventeen of 654 studies were eligible for the meta-analysis. The concentration of airborne microbial measured as aerobic colony count (ACC) was significantly correlated with temperature (r = 0.25 [95% CI = 0.06-0.42], p = 0.01), CO2 concentration (r = 0.53 [95% CI = 0.40-0.64], p Ë‚ 0.001), particle mass concentration (≤5 µg/m3 ; r = 0.40 [95% CI = 0.04-0.66], p = 0.03), and particle size (≤5 and ˃5 µm), (r = 0.51 [95% CI = 0.12-0.77], p = 0.01 and r = 0.55 [95% CI = 0.20-0.78], p = 0.003), respectively, while not being significantly correlated with relative humidity or particulate matter of size >5 µm. Conversely, airborne total fungi (TF) were not significantly correlated with temperature, relative humidity, or CO2 level. However, there was a significant weak correlation between ACC and TF (r = 0.31 [95% CI = 0.07-0.52], p = 0.013). Although significant correlations exist between ACC and IAQ parameters, the relationship is not definitive; the IAQ parameters may affect the microorganisms but are not responsible for the presence of airborne microorganisms. Environmental parameters could be related to the generating source, survival, dispersion, and deposition rate of microorganisms. Future studies should record IAQ parameters and factors such as healthcare worker presence and the activities carried out such as cleaning, sanitizing, and disinfection protocols. Foot traffic would influence both the generation of microorganisms and their deposition rate onto surfaces in the hospital environment. These data would inform models to improve the understanding of the likely concentration of airborne microorganisms and provide an alternative approach for real-time monitoring of the healthcare environment.

Transmission of SARS-CoV-2 by inhalation of respiratory aerosol in the Skagit Valley Chorale superspreading event.

During the 2020 COVID-19 pandemic, an outbreak occurred following attendance of a symptomatic index case at a weekly rehearsal on 10 March of the Skagit Valley Chorale (SVC). After that rehearsal, 53 members of the SVC among 61 in attendance were confirmed or strongly suspected to have contracted COVID-19 and two died. Transmission by the aerosol route is likely; it appears unlikely that either fomite or ballistic droplet transmission could explain a substantial fraction of the cases. It is vital to identify features of cases such as this to better understand the factors that promote superspreading events. Based on a conditional assumption that transmission during this outbreak was dominated by inhalation of respiratory aerosol generated by one index case, we use the available evidence to infer the emission rate of aerosol infectious quanta. We explore how the risk of infection would vary with several influential factors: ventilation rate, duration of event, and deposition onto surfaces. The results indicate a best-estimate emission rate of 970 ± 390 quanta/h. Infection risk would be reduced by a factor of two by increasing the aerosol loss rate to 5 h-1 and shortening the event duration from 2.5 to 1 h.

Evaluation of World Health Organization-Recommended Hand Hygiene Formulations.

As a result of the coronavirus disease pandemic, commercial hand hygiene products have become scarce and World Health Organization (WHO) alcohol-based hand rub formulations containing ethanol or isopropanol are being produced for hospitals worldwide. Neither WHO formulation meets European Norm 12791, the basis for approval as a surgical hand preparation, nor satisfies European Norm 1500, the basis for approval as a hygienic hand rub. We evaluated the efficacy of modified formulations with alcohol concentrations in mass instead of volume percentage and glycerol concentrations of 0.5% instead of 1.45%. Both modified formulations met standard requirements for a 3-minute surgical hand preparation, the usual duration of surgical hand treatment in most hospitals in Europe. Contrary to the originally proposed WHO hand rub formulations, both modified formulations are appropriate for surgical hand preparation after 3 minutes when alcohol concentrations of 80% wt/wt ethanol or 75% wt/wt isopropanol along with reduced glycerol concentration (0.5%) are used.

Bacterial transfer to fingertips during sequential surface contacts with and without gloves.

Bacterial transmission from contaminated surfaces via hand contact plays a critical role in disease spread. However, the fomite-to-finger transfer efficiency of microorganisms during multiple sequential surface contacts with and without gloves has not been formerly investigated. We measured the quantity of Escherichia coli on fingertips of participants after 1-8 sequential contacts with inoculated plastic coupons with and without nitrile gloves. A Bayesian approach was used to develop a mechanistic model of pathogen accretion to examine finger loading as a function of the difference between E coli on surfaces and fingers. We used the model to determine the coefficient of transfer efficiency (λ), and influence of swabbing efficiency and finger area. Results showed that λ for bare skin was higher (49%, 95% CI = 32%-72%) than for gloved hands (30%, CI = 17%-49%). Microbial load tended toward a dynamic equilibrium after four and six contacts for gloved hands and bare skin, respectively. Individual differences between volunteers' hands had a negligible effect compared with use of gloves (P < .01). Gloves reduced loading by 4.7% (CI = -12%-21%) over bare skin contacts, while 20% of participants accrued more microorganisms on gloved hands. This was due to poor fitting, which created a larger finger surface area than bare hands.

Diabetic foot infection: Antibiotic therapy and good practice recommendations.

BACKGROUND: Healthcare events related to diabetic foot disease carry a burden of morbidity, mortality and economic cost. Prompt identification of clinical infection with appropriate tissue sampling limits use of broad spectrum empirical antibiotics and improves antibiotic stewardship. Staphylococcus aureus remains the commonest infecting organism and high-dose flucloxacillin remains the empirical antibiotic of choice for antibiotic naïve patients. Barriers to microbe-specific treatment include: adequate tissue sampling, delays in culture results, drug allergies and the emergence of multidrug-resistant organisms which can complicate the choice of targeted antibiotics. Even appropriate antibiotic treatment carries a risk of adverse events including the selection of resistant organisms. AIMS: Multidisciplinary clinical assessment of a diabetic foot infection is supported by the use of appropriate imaging modalities and deep tissue sampling, both of which are encouraged to enhance sampling accuracy. Narrow-spectrum, high dose, short duration antimicrobial therapy is ideal. Further clarity in these areas would be of benefit to clinicians involved in management of diabetic foot infections. METHODS: A combination of literature review with expert discussion was used to generate consensus on management of diabetic foot infection, with a specific focus on empirical antimicrobial therapy. RESULTS: Gram positive organisms represent the commonest pathogens in diabetic foot infection. However there are developing challenges in antimicrobial resistance and antibiotic availability. DISCUSSION: Recommendations for empirical therapy, including the choice of alternative oral agents and use of outpatient antibiotics would be of benefit to those involved in diabetic foot care. CONCLUSION: This paper provides advice on empirical antibiotic therapy that may be used as a framework for local guideline development to support clinicians in the management of diabetic foot infection.

Dos and don'ts for hospital cleaning.

PURPOSE OF REVIEW: More evidence is emerging on the role of cleaning and decontamination for reducing hospital-acquired infection. Timely and adequate removal of environmental pathogens leads to measurable clinical benefits for patients. This article considers studies published from 2013 examining hospital decontamination technologies and evidence for cost-effectiveness. RECENT FINDINGS: Novel biocides and cleaning products, antimicrobial coatings, monitoring practices and automated equipment are widely accessible. They do not necessarily remove all environmental pathogens, however, and most have yet to be comprehensively assessed against patient outcome. Some studies are confounded by concurrent infection control and/or antimicrobial stewardship initiatives. Few contain data on costs. SUMMARY: As automated dirt removal is assumed to be superior to human effort, there is a danger that traditional cleaning methods are devalued or ignored. Fear of infection encourages use of powerful disinfectants for eliminating real or imagined pathogens in hospitals without appreciating toxicity or cost benefit. Furthermore, efficacy of these agents is compromised without prior removal of organic soil. Microbiocidal activity should be compared and contrasted against physical removal of soil in standardized and controlled studies to understand how best to manage contaminated healthcare environments.

Controlling hospital-acquired infection: focus on the role of the environment and new technologies for decontamination.

There is increasing interest in the role of cleaning for managing hospital-acquired infections (HAI). Pathogens such as vancomycin-resistant enterococci (VRE), methicillin-resistant Staphylococcus aureus (MRSA), multiresistant Gram-negative bacilli, norovirus, and Clostridium difficile persist in the health care environment for days. Both detergent- and disinfectant-based cleaning can help control these pathogens, although difficulties with measuring cleanliness have compromised the quality of published evidence. Traditional cleaning methods are notoriously inefficient for decontamination, and new approaches have been proposed, including disinfectants, steam, automated dispersal systems, and antimicrobial surfaces. These methods are difficult to evaluate for cost-effectiveness because environmental data are not usually modeled against patient outcome. Recent studies have reported the value of physically removing soil using detergent, compared with more expensive (and toxic) disinfectants. Simple cleaning methods should be evaluated against nonmanual disinfection using standardized sampling and surveillance. Given worldwide concern over escalating antimicrobial resistance, it is clear that more studies on health care decontamination are required. Cleaning schedules should be adapted to reflect clinical risk, location, type of site, and hand touch frequency and should be evaluated for cost versus benefit for both routine and outbreak situations. Forthcoming evidence on the role of antimicrobial surfaces could supplement infection prevention strategies for health care environments, including those targeting multidrug-resistant pathogens.

Assessing the impact of a cleaning programme on environmental hygiene in labour and neonatal wards: an exploratory study in The Gambia.

BACKGROUND: Effective surface cleaning in hospitals is crucial to prevent the transmission of pathogens. However, hospitals in low- and middle-income countries face cleaning challenges due to limited resources and inadequate training. METHODS: We assessed the effectiveness of a modified TEACH CLEAN programme for trainers in reducing surface microbiological contamination in the newborn unit of a tertiary referral hospital in The Gambia. We utilised a quasi-experimental design and compared data against those from the labour ward. Direct observations of cleaning practices and key informant interviews were also conducted to clarify the programme's impact. RESULTS: Between July and September 2021 (pre-intervention) and October and December 2021 (post-intervention), weekly surface sampling was performed in the newborn unit and labour ward. The training package was delivered in October 2021, after which their surface microbiological contamination deteriorated in both clinical settings. While some cleaning standards improved, critical aspects such as using fresh cleaning cloths and the one-swipe method did not. Interviews with senior departmental and hospital management staff revealed ongoing challenges in the health system that hindered the ability to improve cleaning practices, including COVID-19, understaffing, disruptions to water supply and shortages of cleaning materials. CONCLUSIONS: Keeping a hospital clean is fundamental to good care, but training hospital cleaning staff in this low-income country neonatal unit failed to reduce surface contamination levels. Further qualitative investigation revealed multiple external factors that challenged any possible impact of the cleaning programme. Further work is needed to address barriers to hospital cleaning in low-income hospitals.

Mandating indoor air quality for public buildings.

If some countries lead by example, standards may increasingly become normalized.

Hospital cleaning: past, present, and future.

INTRODUCTION: The importance of hospital cleaning for controlling healthcare-associated infection (HAI) has taken years to acknowledge. This is mainly because the removal of dirt is inextricably entwined with gender and social status, along with lack of evidence and confusion over HAI definitions. Reducing so-called endogenous infection due to human carriage entails patient screening, decolonisation and/or prophylaxis, whereas adequate ventilation, plumbing and cleaning are needed to reduce exogenous infection. These infection types remain difficult to separate and quantitate. Patients themselves demonstrate wide-ranging vulnerability to infection, which further complicates attempted ranking of control interventions, including cleaning. There has been disproportionate attention towards endogenous infection with less interest in managing environmental reservoirs. QUANTIFYING CLEANING AND CLEANLINESS: Finding evidence for cleaning is compromised by the fact that modelling HAI rates against arbitrary measurements of cleaning/cleanliness requires universal standards and these are not yet established. Furthermore, the distinction between cleaning (soil removal) and cleanliness (soil remaining) is usually overlooked. Tangible bench marking for both cleaning methods and all surface types within different units, with modification according to patient status, would be invaluable for domestic planning, monitoring and specification. AIMS AND OBJECTIVES: This narrative review will focus on recent history and current status of cleaning in hospitals. While its importance is now generally accepted, cleaning practices still need attention in order to determine how, when and where to clean. Renewed interest in removal and monitoring of surface bioburden would help to embed risk-based practice in hospitals across the world.

Risk of organism acquisition from prior room occupants: An updated systematic review.

BACKGROUND: Evidence from a previous systematic review indicates that patients admitted to a room where the previous occupant had a multidrug-resistant bacterial infection resulted in an increased risk of subsequent colonisation and infection with the same organism for the next room occupant. In this paper, we have sought to expand and update this review. METHODS: A systematic review and meta-analysis was undertaken. A search using Medline/PubMed, Cochrane and CINHAL databases was conducted. Risk of bias was assessed by the ROB-2 tool for randomised control studies and ROBIN-I for non-randomised studies. RESULTS: From 5175 identified, 12 papers from 11 studies were included in the review for analysis. From 28,299 patients who were admitted into a room where the prior room occupant had any of the organisms of interest, 651 (2.3%) were shown to acquire the same species of organism. In contrast, 981,865 patients were admitted to a room where the prior occupant did not have an organism of interest, 3818 (0.39%) acquired an organism(s). The pooled acquisition odds ratio (OR) for all the organisms across all studies was 2.45 (95% CI: 1.53-3.93]. There was heterogeneity between the studies (I2 89%, P < 0.001). CONCLUSION: The pooled OR for all the pathogens in this latest review has increased since the original review. Findings from our review provide some evidence to help inform a risk management approach when determining patient room allocation. The risk of pathogen acquisition appears to remain high, supporting the need for continued investment in this area.

Probabilistic microsimulation to examine the cost-effectiveness of hospital admission screening strategies for carbapenemase-producing enterobacteriaceae (CPE) in the United Kingdom.

BACKGROUND: Antimicrobial resistance has been recognised as a global threat with carbapenemase- producing-Enterobacteriaceae (CPE) as a prime example. CPE has similarities to COVID-19 where asymptomatic patients may be colonised representing a source for onward transmission. There are limited treatment options for CPE infection leading to poor outcomes and increased costs. Admission screening can prevent cross-transmission by pre-emptively isolating colonised patients. OBJECTIVE: We assess the relative cost-effectiveness of screening programmes compared with no- screening. METHODS: A microsimulation parameterised with NHS Scotland date was used to model scenarios of the prevalence of CPE colonised patients on admission. Screening strategies were (a) two-step screening involving a clinical risk assessment (CRA) checklist followed by microbiological testing of high-risk patients; and (b) universal screening. Strategies were considered with either culture or polymerase chain reaction (PCR) tests. All costs were reported in 2019 UK pounds with a healthcare system perspective. RESULTS: In the low prevalence scenario, no screening had the highest probability of cost-effectiveness. Among screening strategies, the two CRA screening options were the most likely to be cost-effective. Screening was more likely to be cost-effective than no screening in the prevalence of 1 CPE colonised in 500 admitted patients or more. There was substantial uncertainty with the probabilities rarely exceeding 40% and similar results between strategies. Screening reduced non-isolated bed-days and CPE colonisation. The cost of screening was low in relation to total costs. CONCLUSION: The specificity of the CRA checklist was the parameter with the highest impact on the cost-effectiveness. Further primary data collection is needed to build models with less uncertainty in the parameters.

A Better Disinfectant for Low-Resourced Hospitals? A Multi-Period Cluster Randomised Trial Comparing Hypochlorous Acid with Sodium Hypochlorite in Nigerian Hospitals: The EWASH Trial.

Environmental hygiene in hospitals is a major challenge worldwide. Low-resourced hospitals in African countries continue to rely on sodium hypochlorite (NaOCl) as major disinfectant. However, NaOCl has several limitations such as the need for daily dilution, irritation, and corrosion. Hypochlorous acid (HOCl) is an innovative surface disinfectant produced by saline electrolysis with a much higher safety profile. We assessed non-inferiority of HOCl against standard NaOCl for surface disinfection in two hospitals in Abuja, Nigeria using a double-blind multi-period randomised cross-over study. Microbiological cleanliness [Aerobic Colony Counts (ACC)] was measured using dipslides. We aggregated data at the cluster-period level and fitted a linear regression. Microbiological cleanliness was high for both disinfectant (84.8% HOCl; 87.3% NaOCl). No evidence of a significant difference between the two products was found (RD = 2%, 90%CI: -5.1%-+0.4%; p-value = 0.163). We cannot rule out the possibility of HOCl being inferior by up to 5.1 percentage points and hence we did not strictly meet the non-inferiority margin we set ourselves. However, even a maximum difference of 5.1% in favour of sodium hypochlorite would not suggest there is a clinically relevant difference between the two products. We demonstrated that HOCl and NaOCl have a similar efficacy in achieving microbiological cleanliness, with HOCl acting at a lower concentration. With a better safety profile, and potential applicability across many healthcare uses, HOCl provides an attractive and potentially cost-efficient alternative to sodium hypochlorite in low resource settings.

Insights into the Spectrum of Activity and Mechanism of Action of MGB-BP-3.

MGB-BP-3 is a potential first-in-class antibiotic, a Strathclyde Minor Groove Binder (S-MGB), that has successfully completed Phase IIa clinical trials for the treatment of Clostridioides difficile associated disease. Its precise mechanism of action and the origin of limited activity against Gram-negative pathogens are relatively unknown. Herein, treatment with MGB-BP-3 alone significantly inhibited the bacterial growth of the Gram-positive, but not Gram-negative, bacteria as expected. Synergy assays revealed that inefficient intracellular accumulation, through both permeation and efflux, is the likely reason for lack of Gram-negative activity. MGB-BP-3 has strong interactions with its intracellular target, DNA, in both Gram-negative and Gram-positive bacteria, revealed through ultraviolet-visible (UV-vis) thermal melting and fluorescence intercalator displacement assays. MGB-BP-3 was confirmed to bind to dsDNA as a dimer using nano-electrospray ionization mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Type II bacterial topoisomerase inhibition assays revealed that MGB-BP-3 was able to interfere with the supercoiling action of gyrase and the relaxation and decatenation actions of topoisomerase IV of both Staphylococcus aureus and Escherichia coli. However, no evidence of stabilization of the cleavage complexes was observed, such as for fluoroquinolones, confirmed by a lack of induction of DSBs and the SOS response in E. coli reporter strains. These results highlight additional mechanisms of action of MGB-BP-3, including interference of the action of type II bacterial topoisomerases. While MGB-BP-3's lack of Gram-negative activity was confirmed, and an understanding of this presented, the recognition that MGB-BP-3 can target DNA of Gram-negative organisms will enable further iterations of design to achieve a Gram-negative active S-MGB.