Cefazolin is an essential antibiotic used for the treatment of bacteremia; in particular, it is recommended as a first-line agent for infections caused by methicillin-susceptible Staphylococcus aureus (MSSA). In March 2019, problems with a major antibiotic supplier caused a critical shortage of cefazolin in Japan. In fact, in our hospital, the prescription of cefazolin was restricted (unless permitted by an infectious disease physician) between March 2019 and January 2020. The aim of this study was to evaluate the clinical outcomes of patients with MSSA bacteremia at a university hospital in Japan during cefazolin shortage. Details of antimicrobial use and patient data were extracted from the medical records and classified as pre-shortage (March 2018–January 2019) and post-shortage (March 2019–January 2020). Seventy-five patients were included in the study (pre-shortage group, n = 39; post-shortage group, n = 36); there were no significant differences between the demographic characteristics of the two groups. The percentage of patients that received cefazolin as definitive therapy, was significantly lower in the post-shortage group than that in the pre-shortage group (82% vs. 53%, p = 0.014). Of note, penicillins (including benzylpenicillin, ampicillin, ampicillin/sulbactam, and piperacillin/tazobactam) were more frequently administered as alternative therapy to the post-shortage group (10% vs. 53%, p < 0.001). However, no significant differences were observed between the following clinical outcomes of the two groups: total duration of antibiotic therapy, time to fever resolution and white blood cell normalization, time to negative blood cultures, length of hospitalization after the onset of bacteremia, treatment failure, 30-day mortality, and adverse drug reactions (diarrhea and skin rash). Therefore, in the treatment of MSSA-induced bacteremia, cefazolin shortage was associated with an increase in the use of penicillins as alternative agents, with no major changes in the clinical outcomes.

BACKGROUND: In 2019, according to the Centers for Disease Control and Prevention, Carbpenem-Resistant Enterobacteriaceae (CRE), had cost the lives of over 35,000 patients, particularly the most virulent plasmid-mediated New Delhi metallo-beta-lactamase (NDM). Although healthcare systems normally have strict surveillance and infection control measures for CRE, the rapid emergence of novel SAR-CoV-2 (COVID-19) led to a shortage of personal protective equipment (PPE) and medical supplies. As a result, routine infection practices, such as contact precautions, were violated. Studies have shown this depletion and shift in resources compromised the control of infections such CRE leading to rising horizontal transmission.

METHODS: A retrospective study was conducted at a tertiary healthcare system in Detroit, Michigan to determine the impact of PPE shortages during the COVID-19 pandemic on NDM infection rates. The following time periods were established during 2020 based on PPE availability: (1) pre-PPE shortage (January-June), (2) PPE shortage (July-October), and (3) post-PPE shortage (November-December). Rates of NDM per 10,000 patient days were compared between time periods by Wilcoxon Signed Rank Sum Test. Isolates were confirmed resistant by NDM by molecular typing performed by the Michigan State Health Department. Patient characteristics were gathered by medical chart review and patient interviews by telephone.

RESULTS: Overall, the average rate of NDM infections was 1.82 ±1.5 per 10,000 patient days. Rates during the PPE shortage were significantly higher, averaging 3.6 ±1.1 cases per 10,000 patient days (p = 0.02). During this time, several infections occurred within patients on the same unit and/or patients with same treating team, suggesting possible horizontal transmission. Once PPE stock was replenished and isolation practices were reinstated, NDM infection rates decreased to 0.77 ±1.1 per 10,000 patient days.

CONCLUSION: Control of CRE requires strategic planning with active surveillance, antimicrobial constructs and infection control measures. The study illustrates that in times of crisis, such as COVID-19 pandemic, the burden effective infection control requires much more multidisciplinary efforts to prevent unintentional lapses in patient safety. A swift response by the state and local Health Department at a tertiary healthcare center conveyed a positive mitigation of the highest clinical threats and decreased horizontal transmission of disease.

In recent years, Gram-negative bacterial species such as Acinetobacter baumannii have caused life-threatening infections not treatable with antibiotics. In A. baumannii, the best characterized multidrug efflux system is the prevalent Acinetobacter drug efflux (Ade)ABC tripartite system. The adeA, adeB and adeC genes form an operon, encoding membrane fusion protein, multidrug transporter and outer membrane channel protein structure, respectively. PCR amplification showed that the detection rate of adeB was highest in clinical isolates (Lin, Ling et al. 2009, Choi, Choi et al. 2019). We constructed ten mutated AdeB variants containing single amino acid substitutions in the functionally important regions of AdeB. Purification and protein analyses showed that all mutated AdeB proteins were produced at similar levels. However, cells producing AdeB mutants varied in their susceptibilities to four antibacterials ethidium bromide (Et), gentamicin, zeocin and azithromycin, the known substrates of AdeABC efflux pump. The F178 and D664 residues were identified to be crucial. Cells producing AdeB with D644C showed a rare phenotype: although this mutation reduced the effectiveness of Et efflux, it also had a role in macrolides efflux. F178C enhanced efflux of gentamicin and zeocin as seen from MICs and growth inhibition curves. The concentration- and time-dependent changes of Et fluorescence were detected for all mutants and compared with wild type of AdeB and efflux-deficient hyperporinated cells. Transporter kinetic parameters (Km and B) were analyzed for each mutant. Our results provide a novel insight into the mechanism of AdeB and demonstrate that this transporter is an attractive target for pharmacological development.

Resistance-Nodulation-Division (RND) superfamily of transporters are responsible for the intrinsic antibiotic resistance of Gram-negative bacteria. Widespread nosocomial pathogen Pseudomonas aeruginosa PAO1 strain possesses twelve RND pumps, which are connected with development of clinical multidrug resistance and contribute to virulence, quorum sensing and many other physiological functions. However, the consequences of efflux inhibition on bacterial physiology and how cells might counteract the loss of activities of RND pumps remain unclear. In this study, we use PΔ6 cells carrying deletions in genes encoding the six major RND efflux pumps (mexAB-oprM, mexCD-oprJ, mexJK, mexEF-oprN, mexXY and triABC) in both exponential and stationary phases to analyze how P. aeruginosa changes its physiology in response to the lack of efflux pumps. We compared the transcriptomes of the exponentially growing and stationary PΔ6 and its parent PAO1 cells and identified the functions stressed by the lack of active efflux. This study shows that the mutational inactivation of six major RND pumps elicits a specific transcriptome response, but do not affect the central metabolism. Loss of active efflux generates dramatic changes in transport activities across the cell envelope, cell-to-cell communication and environmental responses, such as iron acquisition, production of pyocyanin, motility or biofilm formation.

The search for producers of biologically active substances that overcome antibiotic-resistance is being actively pursued not only among extremophilic fungi, but also among inhabitants of unique ecological niches with special habitat conditions. These include the intestines of invertebrates: earthworms, millipedes, ants, termites, etc. Fungal spores are found in the stomachs and intestinal tracts of invertebrates. Interaction between fungi and invertebrates contributes to emergence of new compounds that are valuable for further development of antibiotic substances. In this work, we studied fungi associated with larva of black soldier fly Hermetia illucens grown on compound feed for laying hens. Inoculation of the contents of the intestine in the form of a suspension on agar nutrient led to the isolation of a fungal strain with pronounced antimicrobial properties. Phylogenetic analysis based on 18S rRNA revealed that the isolated strain belongs to the genus Byssochlamys. Culture liquid was extracted trice with ethyl acetate; residual antimicrobial activity was controlled by disc-diffusion assay. The culture liquid extract had a broad-spectrum of antimicrobial activity against a number of microorganisms: a fungus (Aspergillus niger INA 00760), a yeast (Candida albicans CBS 8836), gram-positive bacteria (Bacillus subtilis ATCC 6633, Staphylococcus aureus 29213) and gram-negative bacteria (Escherichia coli ATCC 25922). The culture liquid extract was fractionated by flash chromatography and HPLC; the antibiotic was isolated this way. Structure determination of the antibiotic is hampered by its ability to polymerize. Antimicrobial activity of the extract and fractions was monitored with agar well diffusion method and disc-diffusion assay. MIC of the pure substance was measured by the serial dilution method.

The persistence of antibiotic-resistant bacteria in the food chain is an important public health problem. Taking into account the scarcity of available data on fresh vegetables in comparison with those reported on animal products, this study was conducted to determine the importance of vegetables consumed raw as vehicles of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, along with other β-lactamases.

We studied 133 fresh-vegetable samples (lettuce, curly endive, carrot, parsley, tomato, cucumber, pepper and strawberry) and 39 samples from the farm environment (air, water, handler and soil). Enterobacteriaceae and E. coli numbers were counted on CHROMagar Enterobacteria. The presence of antimicrobial-resistant enterobacteria was investigated from overnight streaking a loop from an overnight sample enrichment (Buffered Peptone Water) onto plates of CHROMagar ESBL, CHROMagar KPC, MacConkey agar and MacConkey agar supplemented with cefoxitin (16 µg/ml). Phenotypic confirmation of suspected isolates were performed by disk-based methods and through PCR (bla_CMY-2, bla_CTX-M, bla_TEM, bla_SHV, bla_NDM, bla_OXA-48 and bla_KPC genes).

The highest enterobacteria counts were found on parsley and curly lettuce (5,5±0,3 log cfu/g and 5,5±0,7 log cfu/g, respectively) with no correlation to the detection of antimicrobial-resistant isolates (182). Among them, the detected resistant-genes were bla_CMY-2 (nine strains), bla_CTX-M (three strains), and bla_NDM (two strains). Four strains, isolated from four tomato samples (4/133; 3,0%), were confirmed as ESBL-producers. These strains were identified as Enterobacter cloacae (3) and Serratia fonticola (1) by MALDI-TOF MS. No isolates produced carbapenemases or presented the AmpC-phenotype. Therefore, fresh vegetables could contribute to spread ESBL-producing enterobacteria along the food chain that may have relevance for human health.

Cationic antimicrobials (CA) such as the therapeutic antibiotic colistin (COL) and antiseptics cetrimide (CET) and chlorhexidine (CHX) all exert their mechanism of action by disrupting bacterial membranes, leading to cell content leakage and death. Resistance to CAs is rapidly increasing and of the many antimicrobial resistance mechanisms that may contribute, the role of outer membrane vesicle (OMV) formation is least understood. Here, we gradually adapted E. coli BW215113 K-12 to COL, CET, and CHX by prolonged exposure in vitro to determine how each CA impacts OMV formation. We assessed OMVs isolated from each CA-adapted strain by nanoparticle tracking analysis (NTA) and cryo-transmission electron microscopy (cryo-TEM), and compared OMV proteomes using liquid chromatography-mass spectroscopy. The results of NTA analysis showed that all three CA-adapted strains had increased OMV formation as compared to unadapted BW25113 (WT), where CA-adapted OMVs were significantly larger. Cryo-TEM analyses revealed that each strain’s OMVs had distinctive morphological alterations, where CET-OMVs looked similar to unadapted WT OMVs but were encapsulated and aggregated, CHX-OMVs were multilamellar and COL-OMVs were large (4-12X) and tubular as compared to WT OMVs. Proteomic analysis highlighted significant increases in EptC abundance in COL-OMVs and decreased abundance of MlaA in CHX-OMVs. Antimicrobial susceptibility testing of E. coli BW25113 K-12 supplemented with purified CA-adapted OMVs demonstrated that CET-OMVs did not alter CET susceptibility, CHX-OMVs enhanced BW25113 tolerance to CHX exposure by 2-fold, and COL-OMV supplementation to BW25113 increased COL susceptibility by 2-fold compared to unadapted WT OMVs. Hence, CA adaptation by E. coli has significant ramifications on OMV production and morphology, and CA-adapted OMV exposure has significant consequences for bacterial antimicrobial susceptibility.

Fosfomycin is a first line antibiotic indicated for the treatment of acute uncomplicated cystitis in adult women infected with Escherichia coli or Enterococcus faecalis. Resistance to fosfomycin can be conferred by plasmid-transferable fos genes, which encode modified versions of glutathione transferase enzymes that can inactivate this antibiotic. In Canada, fosfomycin resistance and fos gene detection among E. coli clinical isolates are rare and have not been well described to date. Here, we characterize the fosA genes from three E. coli clinical isolates recovered from Canadian patients (two from urine, one from blood), and two additional fosA genes from the fosA7.5 family. Phylogenetic analysis of these FosA sequences showed close homology to previously characterized FosA proteins. Cloning and overexpression of each fosA sequence in E. coli BW25113 for antimicrobial susceptibility testing confirmed that all fosA genes exhibited fosfomycin-resistant phenotypes (MIC values of >512 and >1024 µg/mL) with the exception of one fosA7.5 variant. Homology modelling of each FosA protein suggests that residue alterations in this inactive fosA7.5 variant may impact fosfomycin drug interactions at the active site region. The findings from this study identify and functionally characterize FosA3, FosA8, as well as novel FosA7.5 members and highlight the importance of phenotypic characterization of fosA genes.

Multidrug antibiotic resistance is a global public health crisis that leads to thousands of people's deaths every year. One of the primary causes of resistance in Gram-negative bacteria such as Escherichia coli is the overexpression of multicomponent molecular machines called multidrug efflux pumps. Efflux pump inhibitors (EPIs)are a promising alternative approach to combat antibiotic resistance. We previously identified a diaminoquinoline acrylamide, NSC-33353, as an active EPI of E. coli efflux pump.

This report will describe a series of cinnamoyl compounds that are analogs of NSC-33353 showing significant activity against the AcrAB-TolC efflux pump of E. coli. To determine the antibacterial properties and propensity of these analogs to act as efflux substrates, we analyzed bacterial growth inhibitory activities of the compounds using efflux proficient and efflux deficient E.coli cells. Surprisingly, the results show that a large number of the analogs possess an antibacterial activity, although their original hit was only a weak antibacterial agent. In the presence of antibiotics novobiocin and erythromycin compounds have a significant potentiation activity. The surface plasmon resonance data show that compounds bind with high affinities both AcrA, a membrane fusion protein, and AcrB, an efflux transporter. The fluorescence-based accumulation assay showed that these compounds inhibit the efflux of fluorescent probes. Taken together, these results show that this series of compounds are promising EPIs.

In summary, we have identified compounds that bind to AcrA, AcrB and potentiate the antibacterial properties of novobiocin and erythromycin in E. coli. We report a new series of EPIs that inhibit the activity of AcrAB-TolC efflux pump.

Evidence has confirmed that excessive use of antibiotics in agricultural farming is the main factor causing antibiotic-resistant (ABR) bacteria such as ABR Staphylococcus aureus, which have a detrimental human health impact. Nevertheless, little effort has given to contain the spread of ABR through animal sources, despite some studies unswervingly reporting a high prevalence of ABR in humans in low and middle-income countries like Bangladesh. This study was aimed to systematically review the studies that reported the prevalence of ABR in S. aureus isolates from animal sources in Bangladesh. Applying the PRISMA guidelines, we searched for studies using the Bangladesh Journal Online, Scopus, PubMed, and EBSCO databases conducted in the last 20 years. The search obtained a total of 124 articles, of which 13 articles (n=1,298) were selected to extract the prevalence data and pooled together. The pooled prevalence of ABR S. aureus was about 49%. The reported data produced a pooled prevalence of ABR (top ten resistant antibiotics) in S. aureus isolates from animal sources (i.e., meat, cow milk, dairy products, eggs, animal feeds and litters) in Bangladesh: streptomycin (90.34%, IQR, interquartile range: 3.67), vancomycin (83.56%, IQR: 16.50), penicillin (79.68 %, IQR: 23.8), Nalidixic acid (78.72 %, IQR: 33), kanamycin (71.10 %, IQR: 28.9), amoxicillin (66.28 %, IQR: 67.86), tetracycline (63.53 %, IQR: 40.44), oxytetracycline (62.86 %, IQR: 11.64), erythromycin (62.28 %, IQR: 41.97), and methicillin (59.50 %, IQR: 40.5). In this systematic review, we demonstrated an evaluation of the evidence of the high prevalence of ABR by S. aureus in animal sources. These findings might have important consequences in policy design to contain the spread of ABR to establish One Health settings in Bangladesh. Additionally, S. aureus is prone to spread ABR among and other bacterial species through mobile genetic elements. Therefore, implementing nationwide surveillance to detect ABR strains and controlling them by establishing strict ABR management is highly recommended.