Association Between Caseload Surge and COVID-19 Survival in 558 U.S. Hospitals, March to August 2020.

BACKGROUND: Several U.S. hospitals had surges in COVID-19 caseload, but their effect on COVID-19 survival rates remains unclear, especially independent of temporal changes in survival. OBJECTIVE: To determine the association between hospitals' severity-weighted COVID-19 caseload and COVID-19 mortality risk and identify effect modifiers of this relationship. DESIGN: Retrospective cohort study. (ClinicalTrials.gov: NCT04688372). SETTING: 558 U.S. hospitals in the Premier Healthcare Database. PARTICIPANTS: Adult COVID-19-coded inpatients admitted from March to August 2020 with discharge dispositions by October 2020. MEASUREMENTS: Each hospital-month was stratified by percentile rank on a surge index (a severity-weighted measure of COVID-19 caseload relative to pre-COVID-19 bed capacity). The effect of surge index on risk-adjusted odds ratio (aOR) of in-hospital mortality or discharge to hospice was calculated using hierarchical modeling; interaction by surge attributes was assessed. RESULTS: Of 144 116 inpatients with COVID-19 at 558 U.S. hospitals, 78 144 (54.2%) were admitted to hospitals in the top surge index decile. Overall, 25 344 (17.6%) died; crude COVID-19 mortality decreased over time across all surge index strata. However, compared with nonsurging (<50th surge index percentile) hospital-months, aORs in the 50th to 75th, 75th to 90th, 90th to 95th, 95th to 99th, and greater than 99th percentiles were 1.11 (95% CI, 1.01 to 1.23), 1.24 (CI, 1.12 to 1.38), 1.42 (CI, 1.27 to 1.60), 1.59 (CI, 1.41 to 1.80), and 2.00 (CI, 1.69 to 2.38), respectively. The surge index was associated with mortality across ward, intensive care unit, and intubated patients. The surge-mortality relationship was stronger in June to August than in March to May (slope difference, 0.10 [CI, 0.033 to 0.16]) despite greater corticosteroid use and more judicious intubation during later and higher-surging months. Nearly 1 in 4 COVID-19 deaths (5868 [CI, 3584 to 8171]; 23.2%) was potentially attributable to hospitals strained by surging caseload. LIMITATION: Residual confounding. CONCLUSION: Despite improvements in COVID-19 survival between March and August 2020, surges in hospital COVID-19 caseload remained detrimental to survival and potentially eroded benefits gained from emerging treatments. Bolstering preventive measures and supporting surging hospitals will save many lives. PRIMARY FUNDING SOURCE: Intramural Research Program of the National Institutes of Health Clinical Center, the National Institute of Allergy and Infectious Diseases, and the National Cancer Institute.

Mycobacterium tuberculosis SatS is a chaperone for the SecA2 protein export pathway.

The SecA2 protein export system is critical for the virulence of Mycobacterium tuberculosis. However, the mechanism of this export pathway remains unclear. Through a screen for suppressors of a secA2 mutant, we identified a new player in the mycobacterial SecA2 pathway that we named SatS for SecA2 (two) Suppressor. In M. tuberculosis, SatS is required for the export of a subset of SecA2 substrates and for growth in macrophages. We further identify a role for SatS as a protein export chaperone. SatS exhibits multiple properties of a chaperone, including the ability to bind to and protect substrates from aggregation. Our structural studies of SatS reveal a distinct combination of a new fold and hydrophobic grooves resembling preprotein-binding sites of the SecB chaperone. These results are significant in better defining a molecular pathway for M. tuberculosis pathogenesis and in expanding our appreciation of the diversity among chaperones and protein export systems.

An orphaned Mce-associated membrane protein of Mycobacterium tuberculosis is a virulence factor that stabilizes Mce transporters.

Mycobacterium tuberculosis proteins that are exported out of the bacterial cytoplasm are ideally positioned to be virulence factors; however, the functions of individual exported proteins remain largely unknown. Previous studies identified Rv0199 as an exported membrane protein of unknown function. Here, we characterized the role of Rv0199 in M. tuberculosis virulence using an aerosol model of murine infection. Rv0199 appears to be a member of a Mce-associated membrane (Mam) protein family leading us to rename it OmamA, for orphaned Mam protein A. Consistent with a role in Mce transport, we showed OmamA is required for cholesterol import, which is a Mce4-dependent process. We further demonstrated a function for OmamA in stabilizing protein components of the Mce1 transporter complex. These results indicate a function of OmamA in multiple Mce transporters and one that may be analogous to the role of VirB8 in stabilizing Type IV secretion systems, as structural similarities between Mam proteins and VirB8 proteins are predicted by the Phyre 2 program. In this study, we provide functional information about OmamA and shed light on the function of Mam family proteins in Mce transporters.

Correlation between GyrA substitutions and ofloxacin, levofloxacin, and moxifloxacin cross-resistance in Mycobacterium tuberculosis.

The newer fluoroquinolones moxifloxacin (MXF) and levofloxacin (LVX) are becoming more common components of tuberculosis (TB) treatment regimens. However, the critical concentrations for testing susceptibility of Mycobacterium tuberculosis to MXF and LVX are not yet well established. Additionally, the degree of cross-resistance between ofloxacin (OFX) and these newer fluoroquinolones has not been thoroughly investigated. In this study, the MICs for MXF and LVX and susceptibility to the critical concentration of OFX were determined using the agar proportion method for 133 isolates of M. tuberculosis. Most isolates resistant to OFX had LVX MICs of >1 µg/ml and MXF MICs of >0.5 µg/ml. The presence of mutations within the gyrA quinolone resistance-determining regions (QRDR) correlated well with increased MICs, and the level of LVX and MXF resistance was dependent on the specific gyrA mutation present. Substitutions Ala90Val, Asp94Ala, and Asp94Tyr resulted in low-level MXF resistance (MICs were >0.5 but ≤2 µg/ml), while other mutations led to MXF MICs of >2 µg/ml. Based on these results, a critical concentration of 1 µg/ml is suggested for LVX and 0.5 µg/ml for MXF drug susceptibility testing by agar proportion with reflex testing for MXF at 2 µg/ml.

Label-free Quantitative Proteomics Reveals a Role for the Mycobacterium tuberculosis SecA2 Pathway in Exporting Solute Binding Proteins and Mce Transporters to the Cell Wall.

Mycobacterium tuberculosis is an example of a bacterial pathogen with a specialized SecA2-dependent protein export system that contributes to its virulence. Our understanding of the mechanistic basis of SecA2-dependent export and the role(s) of the SecA2 pathway in M. tuberculosis pathogenesis has been hindered by our limited knowledge of the proteins exported by the pathway. Here, we set out to identify M. tuberculosis proteins that use the SecA2 pathway for their export from the bacterial cytoplasm to the cell wall. Using label-free quantitative proteomics involving spectral counting, we compared the cell wall and cytoplasmic proteomes of wild type M. tuberculosis to that of a ΔsecA2 mutant. This work revealed a role for the M. tuberculosis SecA2 pathway in the cell wall localization of solute binding proteins that work with ABC transporters to import solutes. Another discovery was a profound effect of SecA2 on the cell wall localization of the Mce1 and Mce4 lipid transporters, which contribute to M. tuberculosis virulence. In addition to the effects on solute binding proteins and Mce transporter export, our label-free quantitative analysis revealed an unexpected relationship between SecA2 and the hypoxia-induced DosR regulon, which is associated with M. tuberculosis latency. Nearly half of the transcriptionally controlled DosR regulon of cytoplasmic proteins were detected at higher levels in the ΔsecA2 mutant versus wild type M. tuberculosis. By increasing the list of M. tuberculosis proteins known to be affected by the SecA2 pathway, this study expands our appreciation of the types of proteins exported by this pathway and guides our understanding of the mechanism of SecA2-dependent protein export in mycobacteria. At the same time, the newly identified SecA2-dependent proteins are helpful for understanding the significance of this pathway to M. tuberculosis virulence and physiology.

Aminoglycoside cross-resistance in Mycobacterium tuberculosis due to mutations in the 5' untranslated region of whiB7.

Since the discovery of streptomycin's bactericidal activity against Mycobacterium tuberculosis, aminoglycosides have been utilized to treat tuberculosis (TB). Today, the aminoglycosides kanamycin and amikacin are used to treat multidrug-resistant (MDR) TB, and resistance to any of the second-line injectable antibiotics, including kanamycin, amikacin, or capreomycin, is a defining characteristic of extensively drug-resistant (XDR) TB. Resistance to kanamycin and streptomycin is thought to be due to the acquisition of unlinked chromosomal mutations. However, we identified eight independent mutations in the 5' untranslated region of the transcriptional activator whiB7 that confer low-level resistance to both aminoglycosides. The mutations lead to 23- to 145-fold increases in whiB7 transcripts and subsequent increased expression of both eis (Rv2416c) and tap (Rv1258c). Increased expression of eis confers kanamycin resistance in these mutants, while increased expression of tap, which encodes an efflux pump, is a previously uncharacterized mechanism of low-level streptomycin resistance. Additionally, high-level resistance to streptomycin arose at a much higher frequency in whiB7 mutants than in a wild-type (WT) strain. Although whiB7 is typically associated with intrinsic antibiotic resistance in M. tuberculosis, these data suggest that mutations in an uncharacterized regulatory region of whiB7 contribute to cross-resistance against clinically used second-line antibiotics. As drug resistance continues to develop and spread, understanding the mechanisms and molecular basis of antibiotic resistance is critical for the development of rapid molecular tests to diagnose drug-resistant TB strains and ultimately for designing regimens to treat drug-resistant cases of TB.

New insights into fluoroquinolone resistance in Mycobacterium tuberculosis: functional genetic analysis of gyrA and gyrB mutations.

Fluoroquinolone antibiotics are among the most potent second-line drugs used for treatment of multidrug-resistant tuberculosis (MDR TB), and resistance to this class of antibiotics is one criterion for defining extensively drug resistant tuberculosis (XDR TB). Fluoroquinolone resistance in Mycobacterium tuberculosis has been associated with modification of the quinolone resistance determining region (QRDR) of gyrA. Recent studies suggest that amino acid substitutions in gyrB may also play a crucial role in resistance, but functional genetic studies of these mutations in M. tuberculosis are lacking. In this study, we examined twenty six mutations in gyrase genes gyrA (seven) and gyrB (nineteen) to determine the clinical relevance and role of these mutations in fluoroquinolone resistance. Transductants or clinical isolates harboring T80A, T80A+A90G, A90G, G247S and A384V gyrA mutations were susceptible to all fluoroquinolones tested. The A74S mutation conferred low-level resistance to moxifloxacin but susceptibility to ciprofloxacin, levofloxacin and ofloxacin, and the A74S+D94G double mutation conferred cross resistance to all the fluoroquinolones tested. Functional genetic analysis and structural modeling of gyrB suggest that M330I, V340L, R485C, D500A, D533A, A543T, A543V and T546M mutations are not sufficient to confer resistance as determined by agar proportion. Only three mutations, N538D, E540V and R485C+T539N, conferred resistance to all four fluoroquinolones in at least one genetic background. The D500H and D500N mutations conferred resistance only to levofloxacin and ofloxacin while N538K and E540D consistently conferred resistance to moxifloxacin only. Transductants and clinical isolates harboring T539N, T539P or N538T+T546M mutations exhibited low-level resistance to moxifloxacin only but not consistently. These findings indicate that certain mutations in gyrB confer fluoroquinolone resistance, but the level and pattern of resistance varies among the different mutations. The results from this study provide support for the inclusion of the QRDR of gyrB in molecular assays used to detect fluoroquinolone resistance in M. tuberculosis.

Microbial activity and diversity in long-term mixed contaminated soils with respect to polyaromatic hydrocarbons and heavy metals.

The co-occurrence of polyaromatic hydrocarbons (PAHs) with heavy metals and their effect on soil microbial activity have not been systematically investigated. In this study a holistic approach was employed by combining physico-chemical, biological and advanced molecular methods to determine the soil microbial activities of long-term mixed contaminated soils collected from a former manufactured gas plant (MGP) site. Concentrations of PAHs in MGP soils ranged from 335 to 8645 mg/kg. Of the potentially toxic metals, concentrations of lead were found to be highest, ranging from 88 to 671 mg/kg, cadmium 8 to 112 mg/kg, while zinc varied from 64 to 488 mg/kg. The enzyme activities were severely inhibited in soils that were contaminated with both PAHs and heavy metals. The presence of heavy metals in PAH-contaminated soils not only reduced the diversity of microbial population but also showed a few distinctive species by exerting selective pressure. The multivariate analysis revealed that there is an association between PAHs and heavy metals which influenced biological properties in mixed contaminated soils. The findings of this study have major implications for the bioremediation of organic pollutants in metal-organic mixed contaminated sites.

Hydrolysis of fenamiphos and its toxic oxidation products by Microbacterium sp. in pure culture and groundwater.

A bacterium with an exceptional ability to hydrolyse fenamiphos and its toxic oxidation products fenamiphos sulfoxide and fenamiphos sulfone, all possessing POC bond was isolated from soil. Based on 16S rRNA gene determination, this bacterium was putatively identified as Microbacterium esteraromaticum. The phenols (fenamiphos phenol, sulfoxide phenol and sulfone phenol) formed during bacterial hydrolysis resisted further degradation in mineral salts medium and sterile groundwater, but were transitory in non-sterile groundwater due to the catabolism of native microorganisms. Also, the cell-free preparation of this bacterium was highly effective in hydrolysing fenamiphos and its oxides. These results demonstrate the potential of this bacterium to detoxify pesticide waste in the environment including the groundwater.

The use of molecular techniques to characterize the microbial communities in contaminated soil and water.

Traditionally, the identification and characterization of microbial communities in contaminated soil and water has previously been limited to those microorganisms that are culturable. The application of molecular techniques to study microbial populations at contaminated sites without the need for culturing has led to the discovery of unique and previously unrecognized microorganisms as well as complex microbial diversity in contaminated soil and water which shows an exciting opportunity for bioremediation strategies. Nucleic acid extraction from contaminated sites and their subsequent amplification by polymerase chain reaction (PCR) has proved extremely useful in assessing the changes in microbial community structure by several microbial community profiling techniques. This review examines the current application of molecular techniques for the characterization of microbial communities in contaminated soil and water. Techniques that identify and quantify microbial population and catabolic genes involved in biodegradation are examined. In addition, methods that directly link microbial phylogeny to its ecological function at contaminated sites as well as high throughput methods for complex microbial community studies are discussed.

Presence and diversity of the beta-lactamase gene in cat and dog staphylococci.

Staphylococci are part of the normal microflora of humans and animals and some are potential pathogens that have become resistant to almost all known antibiotics. Despite the widespread reports of penicillin resistance in cat and dog staphylococci, the mechanism underlying penicillin resistance has not been examined. This study was aimed at investigating the molecular basis of resistance to penicillin in cat and dog staphylococcal isolates that showed phenotypic resistance to beta-lactam antibiotics. An 861 bp fragment of the structural blaZ gene which codes for beta-lactamase production in staphylococci was amplified by polymerase chain reaction (PCR) and the products were sequenced. Sequenced fragments were analysed by protein signature typing and sequences were compared to published blaZ sequences of human and bovine staphylococcal strains held in a public database. Four known protein signature types (1, 3, 5 and 6) and one new type (12) were identified in this study. When sequences were compared with published blaZ sequences, gene phylogenetic analysis revealed three major groups. The four variants of beta-lactamases types (A, B, C and D) belonged to each major group except for types A and D which were both in group II. These findings confirm that the blaZ gene is responsible for beta-lactamase production leading to subsequent resistance to beta-lactam antibiotics in feline and canine staphylococci and that the gene shows similar diversity and relatedness as found with blaZ sequences obtained from human and bovine staphylococci.

Molecular typing of methicillin-resistant staphylococci isolated from cats and dogs.

OBJECTIVES: Methicillin-resistant staphylococci (MRS) isolates from healthy and diseased cats and dogs were characterized by staphylococcal cassette chromosome mec (SCCmec), multilocus sequence typing (MLST) and cassette chromosome recombinase gene (ccrAB) sequencing. METHODS: PCR-directed SCCmec typing was carried out for all MRS isolates and two Staphylococcus aureus and two Staphylococcus epidermidis strains were analysed by MLST. Strains belonging to SCCmec type III and IV were sequenced for their ccrAB gene of allotypes 3 and 2, respectively. RESULTS: Five types of SCCmec, types I, III, IV, IV (paediatric) and V SCCmec, were found. The S. aureus strains belonged to sequence type (ST) 239 and the two S. epidermidis belonged to ST43 and ST60 respectively. High sequence conservation was observed for the ccrAB gene of allotypes 2 and 3. CONCLUSIONS: MRS isolates from cats and dogs demonstrate a similar diversity of SCCmec types to those found in human staphylococci and ST239-MRSA-III, a widely dispersed strain in human hospitals, was identified in diseased dogs.

Partial nucleotide sequencing of the mecA genes of Staphylococcus aureus isolates from cats and dogs.

Methicillin-resistant staphylococci (MRS) pose a challenge to clinicians and health administrators in human medicine, but MRS infections in cats and dogs are not perceived as a problem in veterinary medicine. Ten methicillin-resistant staphylococcal isolates obtained from healthy and diseased cats and dogs were subjected to partial DNA sequencing of the mecA gene. Sequence analysis shows that MRS isolates from both healthy and diseased cats and dogs can harbor the mecA gene. The mecA genes of animal isolates were identical to that found in human MRS strains, and therefore the possibility of zoonotic transfer must be considered.