Kazunori Tomono’s research while affiliated with Osaka Prefectural Institute of Public Health and other places

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Publications (442)


(a) The structure of the micropore module. The micropore module is 25 mm² in size and 0.5 mm thick. The bacterial suspension is introduced from the cis channel, and PBS is introduced from the trans channel. (b) An optical image of the pore of the module. The optical microscopic examination of the micropore module suggests that the 3 μm diameter micropore is in the centre of the silicon substrate. (c) A schema of the micropore. When the bacteria pass through the micropore, the ionic current decreases because of obstruction in flowing ions. The processing software obtains the change in ionic current as a waveform. (d) Scanning electron microscope (SEM) observation of S. epidermidis ×14,000. (e) SEM observation of S. epidermidis ×60,000. (f) SEM observation of S. aureus ×14,000. (g) SEM observation of S. aureus ×60,000. (h) A scatter diagram of the OD600 of the bacterial suspensions and their average pulse counts. No correlations were observed between the OD600 and pulse counts of the bacterial suspensions.
(a) Optical microscope images (scale bar = 10 µm) of S. epidermidis (small dot indicated by yellow arrow) being drawn towards and pulled into a micropore (3 µm black circles): <1> the bacterium near (within 15 µm radius) the micropore is pulled in at a fast rate; <2> the trajectory of a distant bacterium being drawn towards the micropore until it is pulled in. (b) Actual display screen during the measurement. The moment at which the bacteria (yellow arrow) is being sucked into the micropore. Its waveform is denoted on the right top column of the right-side window. The waveforms are indicated at three different time scales using the waveform viewer software.
(a) Histograms of Ip and td of S. epidermidis. (b) Histograms of Ip and td of S. aureus. (c) Differences in the absolute value of the histograms of Ip and td between S. epidermidis and S. aureus. (d) Confusion matrix of all isolates. (e) Confusion matrix of the assembly machine learning results.
(a) Hold-out method. We employed a hold-out method for machine learning that splits data into the following two groups: a training dataset and a testing dataset. (b) Receiver operating characteristic curve of the classifier. (c) Characteristic distribution of the waveform. The pulse data are acquired 250,000 times per min. ‘Steps’ denote the number of data points acquired, ‘Height’ denotes the current value of the pulse, and ‘Peak ratio’ denotes the location of the peak of the pulse, when the left edge of the pulse is 0 and the right edge of the pulse is 1. (d) Zeta potential distribution of the bacteria. The dashed line denotes the median, and the dotted line denotes the quartiles. No statistically significant difference was noted between the two species.
The future perspective of the micropore-artificial intelligence (AI)-based diagnosis. Upon obtaining the clinical samples, they can be analyzed using the modules. The Cloud AI will be able to determine the cause of the infection within a few short minutes.
Using novel micropore technology combined with artificial intelligence to differentiate Staphylococcus aureus and Staphylococcus epidermidis
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March 2024

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45 Reads

Ayumi Morimura

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Hiroyasu Takei

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Methods for identifying bacterial pathogens are broadly categorised into conventional culture-based microbiology, nucleic acid-based tests, and mass spectrometry. The conventional method requires several days to isolate and identify bacteria. Nucleic acid-based tests and mass spectrometry are relatively rapid and reliable, but they require trained technicians. Moreover, mass spectrometry requires expensive equipment. The development of a novel, inexpensive, and simple technique for identifying bacterial pathogens is needed. Through combining micropore technology and assembly machine learning, we developed a novel classifier whose receiver operating characteristic (ROC) curve showed an area under the ROC curve of 0.94, which rapidly differentiated between Staphylococcus aureus and Staphylococcus epidermidis in this proof-of-concept study. Morphologically similar bacteria belonging to an identical genus can be distinguished using our method, which requires no specific training, and may facilitate the diagnosis and treatment of patients with bacterial infections in remote areas and in developing countries.

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Establishment of a modified opsonophagocytic killing assay for anti-pneumococcal surface protein A antibody

August 2023

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8 Reads

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3 Citations

Journal of Microbiological Methods

Streptococcus pneumoniae (pneumococcus) is a pathogenic gram-positive bacterium that causes pneumonia, meningitis, and sepsis. Pneumococcal surface protein A (PspA) induces antibodies that protect against lethal infections by pneumococci. PspA is a choline-binding protein present on the cell surface of almost all pneumococcal strains and is a non-capsular polysaccharide vaccine candidate. For research and development of PspA-based vaccines, an in-vitro test system to measure the activity of functional antibodies capable of killing pneumococci is essential. The opsonophagocytic killing (OPK) assay is used to evaluate the opsonic activity of functional antibodies induced by capsular polysaccharide (CPS)-based vaccines (standard OPK assay). Despite the potential of anti-PspA antibodies to protect against lethal infections in mice, the standard OPK assay fails to evaluate anti-PspA antibodies. Using a pneumococcal surface protein C-deficient strain and extending the incubation time of opsonized bacteria, complement, and HL-60 cells reportedly results in enhanced bactericidal activity (modified OPK assay). We aimed to measure the bactericidal activity of anti-PspA antibodies in intact pneumococcal strains. We optimized the pneumococcal culture method used in the OPK assay to increase the efficiency of anti-PspA antibody-mediated phagocytosis of HL-60 cells. As thick capsules hinder phagocytosis, we attempted to obtain pneumococci with thin capsules through an improved culture method. As pneumococci attached to cells exhibit thin capsules, pneumococci cultured in Todd Hewitt yeast extract (THY) broth were spread on blood agar plates and incubated for 4 h. cpsA mRNA transcript levels in pneumococci cultured on blood agar were lower than those in pneumococci cultured in THY broth. OPK activity against pneumococci expressing PspA of clades 1-5 was reasonably well detected using pneumococci cultured on blood agar in the modified OPK assay. The modified OPK assay for anti-PspA antibody using pneumococci cultured on blood agar represents a useful assay to determine the killing activity of functional anti-PspA antibodies against pneumococci.



Temporal shifts in the predominant carbapenemase gene types among carbapenemase-producing Klebsiella pneumoniae isolated in Bangkok, Thailand, during 2013-2016

June 2023

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58 Reads

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1 Citation

Journal of Medical Microbiology

Introduction. Carbapenemase-producing Enterobacteriaceae (CPE) have emerged as a global threat to public health and clinical practice. Hypothesis/Gap Statement. In Thailand, reports describing CPEs carrying bla NDM and bla OXA-48 -like genes have been increasing recently; however, data on detailed plasmid analysis and temporal shift of sequence type and carbapenemase type are limited. Aim. In this study, we analysed whole-genome sequencing (WGS) data of clinically isolated carbapenemase-producing Klebsiella pneumoniae (CPKP) to reveal the molecular epidemiology of CPKP in a tertiary-care hospital in Bangkok, Thailand. Methodology. Seventy-seven non-duplicated CPKP isolates collected during 2013–2016 were examined for their drug-resistance genes, sequence types and phylogenetic relationships. Results. All the tested isolates possessed carbapenemase gene(s), and the major type of carbapenemase gene in 2014–2015 was bla NDM-1 , whereas isolates in 2016 harboured more bla OXA-232 than bla NDM-1 . Other carbapenemase gene variants, such as bla NDM-4 , bla NDM-5 , bla OXA-48 , bla OXA-181 and bla IMP-14 were detected in some CPKP isolates. Furthermore, this study revealed that CPKP co-harbouring two genes, bla NDM-1 and bla OXA-232 or bla OXA-181 , emerged during this period. Notably, such isolates co-carrying the two carbapenemase genes emerged in three different sequence types, even in a single hospital, and then spread clonally. The WGS of CPKP revealed a temporal shift of the predominant carbapenemase genes from bla NDM-1 to bla OXA-232 along with a variation in other carbapenemase gene types within a span of 4 years. Conclusion. Our findings suggest that a substantial change in CPE types occurred in Thailand and potentially in Southeast Asian countries.


A second update on mapping the human genetic architecture of COVID-19

December 2022

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1,414 Reads

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4 Citations

Investigating the role of host genetic factors in COVID-19 severity and susceptibility can inform our understanding of the underlying biological mechanisms that influence adverse outcomes and drug development. Here we present a second updated genome-wide association study (GWAS) on COVID-19 severity and infection susceptibility to SARS-CoV-2 from the COVID-19 Host Genetic Initiative (data release 7). We performed a meta-analysis of up to 219,692 cases and over 3 million controls, identifying 51 distinct genome-wide significant loci—adding 28 loci from the previous data release. The increased number of candidate genes at the identified loci helped to map three major biological pathways involved in susceptibility and severity: viral entry, airway defense in mucus, and type I interferon.


Fig. 2 Flowchart describing patient selection. All consecutive patients with COVID-19 aged ≥ 18 years who were hospitalized during the study period (between February 2020 and September 2021) and recruited through the Japan COVID-19 Task Force were included. After excluding 160 patients, 3261 patients were enrolled in this study
Clinical characteristics of Japanese COVID-19 patients
Characteristics of hospitalized patients with COVID-19 during the first to fifth waves of infection: a report from the Japan COVID-19 Task Force

December 2022

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254 Reads

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38 Citations

BMC Infectious Diseases

Background: We aimed to elucidate differences in the characteristics of patients with coronavirus disease 2019 (COVID-19) requiring hospitalization in Japan, by COVID-19 waves, from conventional strains to the Delta variant. Methods: We used secondary data from a database and performed a retrospective cohort study that included 3261 patients aged ≥ 18 years enrolled from 78 hospitals that participated in the Japan COVID-19 Task Force between February 2020 and September 2021. Results: Patients hospitalized during the second (mean age, 53.2 years [standard deviation {SD}, ± 18.9]) and fifth (mean age, 50.7 years [SD ± 13.9]) COVID-19 waves had a lower mean age than those hospitalized during the other COVID-19 waves. Patients hospitalized during the first COVID-19 wave had a longer hospital stay (mean, 30.3 days [SD ± 21.5], p < 0.0001), and post-hospitalization complications, such as bacterial infections (21.3%, p < 0.0001), were also noticeable. In addition, there was an increase in the use of drugs such as remdesivir/baricitinib/tocilizumab/steroids during the latter COVID-19 waves. In the fifth COVID-19 wave, patients exhibited a greater number of presenting symptoms, and a higher percentage of patients required oxygen therapy at the time of admission. However, the percentage of patients requiring invasive mechanical ventilation was the highest in the first COVID-19 wave and the mortality rate was the highest in the third COVID-19 wave. Conclusions: We identified differences in clinical characteristics of hospitalized patients with COVID-19 in each COVID-19 wave up to the fifth COVID-19 wave in Japan. The fifth COVID-19 wave was associated with greater disease severity on admission, the third COVID-19 wave had the highest mortality rate, and the first COVID-19 wave had the highest percentage of patients requiring mechanical ventilation.


Replicon types of representative plasmids
A Nationwide Plasmidome Surveillance in Thailand Reveals a Limited Variety of New Delhi Metallo-β-Lactamase-Producing Carbapenem-Resistant Enterobacteriaceae Clones and Spreading Plasmids

November 2022

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55 Reads

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6 Citations

Despite frequent identification of plasmids carrying carbapenemase genes, the transfer of plasmids carrying carbapenemase genes is not well recognized in clinical settings because of technical limitations. To investigate the detailed mechanisms of the spread of carbapenem-resistant Enterobacteriaceae (CRE), we performed multifaceted genomic surveillance of CRE isolates in Thailand and analyzed their plasmidome. We analyzed 371 Enterobacteriaceae isolates carrying blaNDM-1 and 114 Enterobacteriaceae isolates carrying blaNDM-5 obtained from clinical samples of 473 patients in 11 representative hospitals located in six provinces in Thailand between 2012 and 2017. The complete structures of plasmids carrying blaNDM and chromosomal phylogeny were determined by combining Southern blotting hybridization analysis and our previously performed whole-genome short-read sequencing data. Dissemination of the blaNDM-5 gene among the Enterobacteriaceae isolates in Thailand was mainly owing to the nationwide clonal spread of Escherichia coli ST410 and regional clonal spreads of Escherichia coli ST361 and ST405. Analysis of blaNDM-1-carrying isolates revealed nationwide dissemination of two specific plasmids and nationwide clonal dissemination of Klebsiella pneumoniae ST16 accompanied with regional disseminations of three distinctive K. pneumoniae clones (ST231, ST14, and ST147) with different plasmids. Dissemination of CRE carrying blaNDM in Thailand is mainly based on nationwide clonal expansions of E. coli ST410 carrying blaNDM-5 and K. pneumoniae ST16 carrying blaNDM-1, nationwide dissemination of two distinctive plasmids carrying blaNDM-1, and accumulation of clonal expansions in regional areas. Although the overuse of antibiotics can promote CRE dissemination, the limited variety of transmitters highlights the importance of preventing horizontal dissemination among patients.


Establishment and clinical application of SARS-CoV-2 catch column

November 2022

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25 Reads

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2 Citations

Clinical and Experimental Nephrology

Background: A certain number of patients with coronavirus disease 2019 (COVID-19), particularly those who test positive for SARS-CoV-2 in the serum, are hospitalized. Further, some even die. We examined the effect of blood adsorption therapy using columns that can eliminate SARS-CoV-2 on the improvement of the prognosis of severe COVID-19 patients. Methods: This study enrolled seven patients receiving mechanical ventilation. The patients received viral adsorption therapy using SARS-catch column for 3 days. The SARS-catch column was developed by immobilizing a specific peptide, designed based on the sequence of human angiotensin-converting enzyme 2 (hACE2), to an endotoxin adsorption column (PMX). In total, eight types of SARS-CoV-2-catch (SCC) candidate peptides were developed. Then, a clinical study on the effects of blood adsorption therapy using the SARS-catch column in patients with severe COVID-19 was performed, and the data in the present study were compared with historical data of severe COVID-19 patients. Results: Among all SCC candidate peptides, SCC-4N had the best adsorption activity against SARS-CoV-2. The SARS-catch column using SCC-4N removed 65% more SARS-CoV-2 than PMX. Compared with historical data, the weaning time from mechanical ventilation was faster in the present study. In addition, the rate of negative blood viral load in the present study was higher than that in the historical data. Conclusion: The timely treatment with virus adsorption therapy may eliminate serum SARS-CoV-2 and improve the prognosis of patients with severe COVID-19. However, large-scale studies must be performed in the future to further assess the finding of this study (jRCTs052200134).


Basic characteristics.
GMT of SARS-CoV-2 S glycoprotein-specific antibody.
Cellular response (IFN-γ) to AG0302.
Phase I Study to Assess the Safety and Immunogenicity of an Intradermal COVID-19 DNA Vaccine Administered Using a Pyro-Drive Jet Injector in Healthy Adults

August 2022

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27 Reads

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10 Citations

We conducted a nonrandomized, open-label phase I study to assess the safety and immunogenicity of an intradermal coronavirus disease 2019 (COVID-19) DNA vaccine (AG0302-COVID-19) administered using a pyro-drive jet injector at Osaka University Hospital between Yanagida November 2020 and December 2021. Twenty healthy volunteers, male or female, were enrolled in the low-dose (0.2 mg) or high-dose (0.4 mg) groups and administered AG0302-COVID19 twice at a 2-week interval. There were no adverse events that led to discontinuation of the study drug vaccination schedule. A serious adverse event (disc protrusion) was reported in one patient in the high-dose group, but the individual recovered, and the adverse event was not causally related to the study drug. In the analysis of the humoral immune response, the geometric mean titer (GMT) of serum anti-SARS-CoV-2 spike glycoprotein-specific antibody was low in both the low-dose and high-dose groups (246.2 (95% CI 176.2 to 344.1, 348.2 (95% CI 181.3 to 668.9)) at the 8 weeks after first vaccination. Regarding the analysis of the cellular immune, the number of IFN-γ-producing cells responsive to the SARS-CoV-2 spike glycoprotein increased with individual differences after the first dose and was sustained for several months. Overall, no notable safety issues were observed with the intradermal inoculations of AG0302-COVID19. Regarding immunogenicity, a cellular immune response was observed in some subjects after AG0302-COVID19 intradermal inoculation, but no significant antibody production was observed.


The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

August 2022

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381 Reads

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18 Citations

Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection. Genetic mechanisms influencing COVID-19 susceptibility are not well understood. Here, the authors analyzed whole blood RNA-seq data of 465 Japanese individuals with COVID-19, highlighting thousands of fine-mapped variants affecting expression and splicing of genes, as well as the presence of COVID-19 severity-interaction eQTLs.


Citations (79)


... The copyright holder for this preprint this version posted November 30, 2024. ; https://doi.org/10.1101/2024.11.29.626107 doi: bioRxiv preprint Streptococcus pneumoniae (32)and has been suggested as a potential protective activity against Neisseria infections, this activity also did not have a correlation with N. gonorrhoeae clearance in OMV immunized mice (33,34). Whether there are other antibody-mediated mechanisms of protection that we did not measure remains a possibility. ...

Reference:

Protection against N. gonorrhoeae induced by OMV-based Meningococcal Vaccines are associated with cross-species directed humoral and cellular immune responses
Establishment of a modified opsonophagocytic killing assay for anti-pneumococcal surface protein A antibody
  • Citing Article
  • August 2023

Journal of Microbiological Methods

... We also re-estimated and leveraged the genetic correlation between the traits to amplify statistical power to discover IPF associations by performing multi-trait meta-analysis (MTAG) 24 . For these analyses we used the IPF meta-analysis statistics produced in this study and the latest meta-analysis summaries (Freeze 7) from the COVID-19 Host Genetics Initiative (HGI), for the Hospitalised covid vs. population phenotype (B2_ALL_leave_23andme), as this has produced the best balance between a carefully curated phenotype for severity and statistical power 15,25 (Supplementary Table 15). ...

A second update on mapping the human genetic architecture of COVID-19

... This threat has been evident since the discovery of plasmidmediated mcr-1 in E. coli isolated from humans and pigs in China in 2015 (Liu et al., 2016), when the effectiveness of antibiotics was significantly reduced. Many studies have reported the coexistence of mcr-1 and bla NDM (Cao et al., 2021;Abe et al., 2022;Kuang et al., 2022;Wang et al., 2023). In addition, in this study, we conducted computational statistical analyses to examine correlations among ARGs as well as between AMR phenotypes and ARGs. ...

A Nationwide Plasmidome Surveillance in Thailand Reveals a Limited Variety of New Delhi Metallo-β-Lactamase-Producing Carbapenem-Resistant Enterobacteriaceae Clones and Spreading Plasmids

... SARS-CoV-2 binds to angiotensin-converting enzyme 2 (ACE2) for cellular entry, while viral neutralization by soluble ACE2 or antibodies can block the systemic spread [6,7]. A blood adsorption column, which can capture SARS-CoV-2 by a part of ACE2 peptide immobilized on the surface, showed the potential to improve the prognosis of patients with severe COVID-19 requiring artificial ventilation [8]. The exploration of biomarkers has led to the discovery of therapeutic options. ...

Establishment and clinical application of SARS-CoV-2 catch column
  • Citing Article
  • November 2022

Clinical and Experimental Nephrology

... The electroporation conditions were bridged to a clinically used electroacupuncture machine and for clinical trials [68]. In addition to electroporation, several alternative delivery systems have been developed, such as microneedle arrays [69], jet injectors [70][71][72][73], the gene gun method [74][75][76], and suction-based devices [77][78][79][80]. However, the efficacy still needs to be improved. ...

Phase I Study to Assess the Safety and Immunogenicity of an Intradermal COVID-19 DNA Vaccine Administered Using a Pyro-Drive Jet Injector in Healthy Adults

... In this study, we systematically characterized shared versus mRNA expression or protein-specific QTLs using a collection of 1,405 genotyped samples, whole-blood mRNA and 2,932 plasma protein expression data (measured using Olink Explore 3072) in the Japan COVID- 19 Task Force ( JCTF 20,21 ), including 998 samples and 2,211 genes with both mRNA and protein measurements (Extended Data Fig. 1). Using statistical fine-mapping, we describe the distinctive features for each class of QTLs, show the relevance of protein-specific QTLs to complex traits and provide examples where protein-specific QTLs explain variant-disease associations. ...

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

... Here, we identified a carbapenemase-producing K. pneumoniae ST16 isolate in Switzerland. This lineage, considered an emerging high-risk clone, has been increasingly reported worldwide and has been associated with severe clinical outcomes [25][26][27]. Overall, while WGS is increasingly used for Gram-negative epidemiological surveillance [28], comparing the pros and cons of each sequencing technique becomes paramount. Long-read-based surveillance allowing genomic architecture assessment and plasmid characterization is increasingly necessary [29]. ...

Clonal dissemination of carbapenem-resistant Klebsiella pneumoniae ST16 co-producing NDM-1 and OXA-232 in Thailand

JAC-Antimicrobial Resistance

... DOCK2 activates proinflammatory macrophages to promote the expression of pro-inflammatory genes [15]. Targeting DOCK2 impairs pulmonary macrophage recruitment and infiltration [14,16], as well as reduces RAC1 activation and reactive oxygen species (ROS) production [17]. In addition, DOCK2 facilitates the transition from lung fibroblast to myofibroblast, thereby promoting pulmonary fibrosis [18]. ...

DOCK2 is involved in the host genetics and biology of severe COVID-19

Nature

... Moreover, the biofilm matrix, rich in proteins and eDNA, enhances resistance by adhering to antibiotics and diminishing their effectiveness. This composition protects bacterial cells and reduces the efficacy of antibiotics specifically formulated with anti-biofilm characteristics [118]. ...

Enhancement ofAcinetobacterbaumanniibiofilm growth by cephem antibiotics via enrichment of protein and extracellular DNAin thebiofilm matrices

Journal of Applied Microbiology