Biofilm Resistance Research Articles

Antibiotic susceptibility and biofilm formation in multidrug resistant Pseudomonas aeruginosa

one of the Gram-Negative pathogens, serves a crucial organism in nosocomial outbreaks. The properties of this bacteria including adaptability, biofilm formation, etc. make this organism develop resistance against various antibiotic drugs. The current study aims to screen the multidrug resistant isolates for biofilm formation and to study the relationship of drug resistance and development of biofilm. Clinical samples growing in culture were screened for antibiotic susceptibility pattern using disc diffusion method. The multidrug resistant isolates of identified using culture and standard microbiological tests were included in the study and were tested for biofilm formation using micro titre plate assay: Out of the 224 clinical samples growing in culture, 100 isolates were found to be multidrug resistant (MDR). 42% of the isolates showed resistance to Cefepime followed by 26% isolates were resistant to Levofloxacin. 88% of the MDR isolates produced biofilm and among these 75% produced strong biofilm, 10% of the isolates produced weak biofilm and 3% of the isolates produced moderate biofilms. The present study observed that majority of the MDR isolates were found to be biofilm producers. Thus, biofilm production is said to be one of the important properties of the organism which could be attributed to their multi drug resistance.

Characterization of a carbapenemase-producing Klebsiella pneumoniae isolate of a patient in an intensive care unit in Greece: A study of resistome, virulome, and mobilome.

Klebsiella pneumoniae is a major pathogen associated with hospital-acquired infections, particularly those involving multidrug-resistant strains. Carbapenem resistance, often driven by carbapenemases such as KPC, VIM, OXA-48, and NDM, poses a significant challenge in clinical settings. This study reports on K. pneumoniae strain A165, isolated from a blood culture of a 51-year-old female patient hospitalized for respiratory distress post-SARS-CoV-2 infection. This K. pneumoniae strain exhibited resistance to several antibiotics, including carbapenems, cephalosporins, aminoglycosides, and fluoroquinolones, but remained susceptible to gentamicin, colistin, and trimethoprim-sulfamethoxazole. Next-generation sequencing was performed on Ion torrent platform, that revealed a genome size of 5,676,404 bp, including a chromosome and six plasmids. The strain was classified as sequence type 11 (ST11), a high-risk lineage associated with carbapenem resistance. The resistome of A165 included multiple β-lactamase genes, such as blaNDM-1 and blaOXA-48, as well as genes conferring resistance to other antibiotic classes. The virulome analysis identified genes involved in iron acquisition (yersiniabactin operon genes: ybtE, ybtT, irp1, irp2; aerobactin receptor: iutA), adhesion (mrkA-J, fimA-K), capsule and biofilm formation (rcsA, rcsB, ompA) and resistance to complement (traT) contributing to its pathogenic potential. The mobilome analysis revealed nine insertion sequences, including ISKpn1, ISKpn18, ISKpn43, ISKpn28, ISKpn14, ISEcp1, and IS6100. The strain also harbored six replicons: Col440II, ColRNAI, IncFIA(HI1), IncFIB(K), IncFII(K), and IncR, which are associated with the horizontal transfer of resistance and virulence genes. Comparative analysis with global isolates demonstrated the widespread dissemination of carbapenemase-producing K. pneumoniae, with notable occurrences in Europe, Asia, and the Americas. This study highlights the growing concern of multidrug-resistant K. pneumoniae in hospital settings and emphasizes the need for robust surveillance and infection control measures.

Serum amikacin concentrations in dogs with naturally occurring open wounds treated with topical amikacin in carboxymethylcellulose hydrogel.

To quantify serum amikacin concentrations in dogs undergoing wound management with topical amikacin (45 mg/mL) 3% carboxymethylcellulose hydrogel. Prospective clinical study. Eleven client-owned dogs. Dogs with naturally occurring wounds, undergoing treatment with topical amikacin gel, were enrolled. A whole blood sample was collected prior to initial application of the gel. Up to a maximum dose of 30 mg/kg of gel, was applied directly on the wound and the wound was bandaged. Serial blood sampling was performed at approximately 2, 4, 8, 12, 18, 24, 32, 40, 48, 56, 64, and 72 h after application of amikacin gel. The sampling schedule was reset following each bandage change and new application of the gel. Up to 20 samples per dog were collected. The Siemens Syva EMIT Amikacin Assay was used to quantify the concentration of amikacin in each blood serum sample. The lower limit of quantification (LLOQ) of the test was 2.5 μg/mL. Amikacin gel was applied a total of 31 times (dose range, 0.1-24.9 mg/kg). A total of 153 samples were analyzed. Five samples in three different dogs were above the LLOQ at approximately 2 h after gel application (range 2.75-3.82 μg/mL). All other samples were below the LLOQ. Routine use of amikacin gel for open wound management did not result in serum amikacin levels above 5 μg/mL. Topical amikacin gel may be a safe treatment option for wounds in dogs with resistant infections or biofilms.

Citrus flavonoids diosmin, myricetin and neohesperidin as inhibitors of Pseudomonas aeruginosa: Evidence from antibiofilm, gene expression and in vivo analysis.

Citrus flavonoids are group of bioactive polyphenols. Here, we investigated the potential of diosmin, myricetin and neohesperidin as possible inhibitors of Pseudomonas aeruginosa. This bacterium is a major clinical challenge due to its propensity to form resistant biofilm. The aims of this study were to examine flavonoids antibacterial activity using the microdilution method, assays intended to determine several antibiofilm mechanisms (crystal violet, congo red binding, extracellular DNA (eDNA) test and confocal laser scanning microscopy (CLSM) live/dead cell imaging), followed by virulence genes RT-qPCR analysis. Furthermore, we aimed to examine in vivo toxicity of the compounds as well as their efficacy in P. aeruginosa zebrafish embryo infection model. Minimal inhibitory concentrations of tested flavonoids towards P. aeruginosa were in range 0.05 - 0.4 mg/mL. A high potential of the compounds to disturb both the formation of the bacterial biofilm and its eradication was recorded, including significant reduction in biofilm biomass, exopolysaccharide and eDNA production. Biofilm treatment with diosmin resulted in the lowest percentage of live microbial cells as observed in the CLSM live/dead cell imaging. The lasI, pvdS, and rhlC genes were found to be downregulated in the presence of diosmin and myricetin. Only diosmin stood out as non-embryotoxic. Consequently, in vivo analysis using a zebrafish model of P. aeruginosa infection showed an antivirulence effect of diosmin. Our findings suggest that diosmin could be potential candidate for the development of new agent that target P. aeruginosa infections by reducing its virulence mechanisms.

The global regulator SpoVG is involved in biofilm formation and stress response in foodborne Staphylococcus aureus

Staphylococcus aureus (S. aureus) is a primary culprit of food poisoning. As a highly adaptable pathogen, S. aureus demonstrates formidable biofilm-forming and stress tolerance capabilities, inducing significant challenges to eradicate food contamination caused by this organism. SpoVG, a regulatory protein in S. aureus, controls the expression of numerous genes. However, its role in biofilm formation and stress response in foodborne S. aureus remains to be elucidated. In this study, we investigated the functions of SpoVG involved in food-related stress responses and biofilm formation in S. aureus RMSA50. The results demonstrated that SpoVG deletion enhanced biofilm formation and resistance to heat and desiccation, while decreased tolerance to oxidative stress. Further analysis revealed that cell aggregation and the accumulation of extracellular DNA (eDNA) may contribute to the enhanced biofilm formation. Real-time quantitative reverse transcription-PCR (RT-qPCR) revealed that the expression levels of nuc and sasC, which are related to cell aggregation and eDNA concentration, were significantly altered in the spoVG mutant. Electrophoretic mobility shift assays (EMSA) confirmed that SpoVG directly binds to the promoter region of nuc and sasC to regulate their expression. These findings suggest that SpoVG may serve as a target to decrease biofilm formation and control S. aureus contamination in the food industry.

Characterization of Pseudomonas aeruginosa Isolated from Bovine Mastitis in Northern Jiangsu Province and Correlation to Drug Resistance and Biofilm Formability.

This study aimed to provide experimental support for the prevention and treatment of Pseudomonas aeruginosa infections and to elucidate the epidemiological distribution of resistance and virulence genes of Pseudomonas aeruginosa from mastitis in dairy cows in the northern part of Jiangsu Province and their relationship with the biofilm-forming ability of the strains. Mastitis presents a significant challenge within dairy farming, adversely impacting the health of dairy cows and precipitating substantial economic losses in milk production. In this study, Pseudomonas aeruginosa (PA) was isolated and identified from mastitis milk samples in Jiangsu Province, China. In order to characterize the isolates, multilocus sequence typing (MLST), drug resistance phenotypes, virulence genes, and biofilm formations were detected. The isolation and identification of pathogenic bacteria from 168 clinical mastitis milk samples using 16S rRNA and PCR revealed 63 strains of Pseudomonas aeruginosa, which were determined to be highly homologous according to phylogenetic tree analysis. In addition, the MLST indicated five major ST types, namely ST277, ST450, ST571, ST641, and ST463. The susceptibility to 10 antimicrobials was determined, and it was found that 63 strains of Pseudomonas aeruginosa did not have a strong resistance to the antimicrobials in general. However, there were differences in the phenotypes' resistance to antimicrobials among the different ST types. It was also found that the more resistant the strains were to antimicrobials, the lower the carriage of virulence genes detected. The biofilm content was measured using the semi-quantitative crystal violet method. It was found that there were a few strains with medium or strong biofilm-forming abilities. However, the number of virulence genes carried by the 63 strains of Pseudomonas aeruginosa was inversely proportional to the biofilm-forming ability. It was also found that there were significantly more Pseudomonas aeruginosa in the biofilm state than in the planktonic state and that strains with strong biofilm-forming abilities were more resistant to antimicrobials.

Comprehensive Study of Antibiotic Resistance in Enterococcus spp.: Comparison of Influents and Effluents of Wastewater Treatment Plants.

Background/Objectives: The spread of antibiotic resistance, particularly through Enterococcus spp., in wastewater treatment plants (WWTPs) poses significant public health risks. Given that research on antibiotic-resistant enterococci and their antibiotic-resistance genes in aquatic environments is limited, we evaluated the role of Enterococcus spp. in WWTPs by comparing the antibiotic resistance rates, gene prevalence, biofilm formation, and residual antibiotics in the influent and effluent using culture-based methods. Methods: In 2022, influent and effluent samples were collected from 11 WWTPs in South Korea. Overall, 804 Enterococcus strains were isolated, and their resistance to 16 antibiotics was assessed using the microdilution method. Results: High resistance to tetracycline, ciprofloxacin, kanamycin, and erythromycin was observed. However, no significant differences in the overall resistance rates and biofilm formation were observed between the influent and effluent. Rates of resistance to ampicillin, ciprofloxacin, and gentamicin, as well as the prevalence of the tetM and qnrS genes, increased in the effluent, whereas resistance rates to chloramphenicol, florfenicol, erythromycin, and tylosin tartrate, along with the prevalence of the optrA gene, decreased. E. faecium, E. hirae, and E. faecalis were the dominant species, with E. faecalis exhibiting the highest resistance. Conclusions: Our results suggest that WWTPs do not effectively reduce the rates of resistant Enterococcus spp., indicating the need for continuous monitoring and improvement of the treatment process to mitigate the environmental release of antibiotic-resistant bacteria.

Efflux pumps: gatekeepers of antibiotic resistance in Staphylococcus aureus biofilms.

Staphylococcus aureus, a versatile human pathogen, poses a significant challenge in healthcare settings due to its ability to develop antibiotic resistance and form robust biofilms. Understanding the intricate mechanisms underlying the antibiotic resistance is crucial for effective infection treatment and control. This comprehensive review delves into the multifaceted roles of efflux pumps in S. aureus, with a focus on their contribution to antibiotic resistance and biofilm formation. Efflux pumps, integral components of the bacterial cell membrane, are responsible for expelling a wide range of toxic substances, including antibiotics, from bacterial cells. By actively extruding antibiotics, these pumps reduce intracellular drug concentrations, rendering antibiotics less effective. Moreover, efflux pumps have emerged as significant contributors to both antibiotic resistance and biofilm formation in S. aureus. Biofilms, structured communities of bacterial cells embedded in a protective matrix, enable S. aureus to adhere to surfaces, evade host immune responses, and resist antibiotic therapy. Efflux pumps play a pivotal role in the development and maintenance of S. aureus biofilms. However, the interplay between efflux pumps, antibiotic resistance and biofilm formation remains unexplored in S. aureus. This review aims to elucidate the complex relationship between efflux pumps, antibiotic resistance and biofilm formation in S. aureus with the aim to aid in the development of potential therapeutic targets for combating S. aureus infections, especially those associated with biofilms. The insights provided herein may contribute to the advancement of novel strategies to overcome antibiotic resistance and disrupt biofilm formation in this clinically significant pathogen.

A Mucous Permeable Local Delivery Strategy Based on Manganese-Enhanced Bacterial Cuproptosis-like Death for Bacterial Pneumonia Treatment.

Bacterial pneumonia is one of the most challenging global infectious diseases with high morbidity and mortality. Considering the antibiotic abuse and resistance of bacterial biofilms, a variety of metal-based materials have been developed. However, due to the high oxygen environment of the lungs, some aerobic infection bacteria have high tolerance to oxygen and ROS, and most of the metal-based materials based on ROS may not achieve good therapeutic effects. Inspired by the sensitivity of cuproptosis to aerobic respiratory cells, we designed a copper composite antibacterial nanoparticle and found that it can effectively induce cuproptosis-like death in the aerobic bacteria of the lungs. To address the challenge of in vivo application of cuproptosis, manganese dioxide was first incorporated to deplete protective glutathione, which can interact with copper and thus hinder the interaction of copper with proteins and assist in antibacterial action through immune enhancement. Cuproptosis-like death also requires a large number of copper ions. To meet this demand, we deliver positively hydrophilic modified composite nanoparticles that effectively penetrate the lung mucus layer directly to the lungs through local administration, and the copper ions are further released rapidly by the acidic environment at the infected site, which can further destroy bacterial biofilms in synergy with manganese. This drug-delivery system can effectively treat pneumonia caused by aerobic bacteria and avoid systemic toxicity that can be caused by large doses of copper.

Clinicopathological findings and etiological characterization in caprine gangrenous mastitis

Gangrenous mastitis ranks among the most economically significant diseases affecting small ruminants. In the present study, along with clinicopathological studies, the etiological characterisation and the contribution of various virulence factors of caprine gangrenous mastitis was studied. Staphylococcus aureus with colony count ranging from 106 to 108 CFU/mL has been identified as the etiological agent in 15 gangrenous mastitis affected goats based on cultural isolation, biochemical characteristics and molecular confirmation. Haematological and serum biochemical analysis was carried out in the affected goats which revealed leucocytosis with thrombocytopenia and hypoproteinemia. Phenotypic identification of antibiotic resistance revealed significantly high resistance to enrofloxacin, sulpha-trimethoprim, tetracycline, and gentamicin whereas the antibiotics cefoperazone, ceftriaxone- tazobactam and amoxicillin clavulanate showed least resistance. Toxin genes lukMF, and hla were detected in 66.7 per cent and 60 per cent of the isolates respectively. Antibiotic resistant genes blaZ (13.3 %), tetM (20 %), sul1 (13.3 %), mecA (80 %) and biofilm forming genes like icaA (26.6 %) icaD (40 %) were also detected in the S. aureus isolates. Majority of the isolates revealed significantly higher antibiotic resistance along with toxin genes and all the isolates showed biofilm formation. Biofilm forming ability of S. aureus plays a crucial role in its virulence whereas, antimicrobial resistance has a significant role in potentiating the virulence of pathogen.

Mechanism and nanotechnological-based therapeutics for tolerance and resistance of bacterial biofilms.

Bacterial biofilms are one of the most relevant drivers of chronic and recurrent infections and a significant healthcare problem. Biofilms were formed by cross-linking of hydrophobic extracellular polymeric substances (EPS), such as proteins, polysaccharides, and eDNA, which were synthesized by bacteria themselves after adhesion and colonization on biological surfaces. They had the characteristics of dense structure and low drug permeability, leading to tolerance and resistance of biofilms to antibiotics and to host responses. Within a biofilm, microbial cells show increased tolerance to both immune system defense mechanisms and antimicrobials than the same cells in the planktonic state. It is one of the key reasons for the failure of traditional clinical drug to treat infectious diseases. Currently, no drugs are available to attack bacterial biofilms in the clinical setting. The development of novel preventive and therapeutic strategies is urgently needed to allow an effective management of biofilm-associated infections. Based on the properties of nanomaterials and biocompatibility, nanotechnology had the advantages of specific targeting, intelligent delivery and low toxicity, which could realize efficient intervention and precise treatment of biofilm-associated infections. In this paper, the mechanisms of bacterial biofilm resistance to antibiotics and host response tolerance were elaborated. Meanwhile, This paper highlighted multiple strategies of biofilms eradication based on nanotechnology. Nanotechnology can interfere with biofilm formation by destroying mature biofilm, modulating biofilm heterogeneity, inhibiting bacterial metabolism, playing antimicrobial properties, activating immunity and enhancing biofilm penetration, which is an important new anti-biofilm preparation. In addition, we presented the key challenges still faced by nanotechnology in combating bacterial biofilm infections. Utilization of nanotechnology safely and effectively should be further strengthened to confirm the safety aspects of their clinical application.

Enhancement of stress resistance of electroactive biofilms against hypersaline shock via exogenous electron mediator addition

Unexpected hypersaline shock from saline wastewater poses a common challenge in the biological treatment processes of WWPTs. Bioelectrochemical systems (BESs) possess an inherent advantage for treating hypersaline wastewater. The elevated salinity enhances the wastewater’s conductivity, which in turn significantly accelerates the electron and proton transfer within the system. However, excessive increases in salinity can impede electron transport capacity, potentially resulting in cell plasmolysis and system failure in severe instances. Here, we investigated the impact of electron mediator (EM) addition on the resilience of electroactive biofilms (EB) to hypersaline environments in BESs. The EB developed under anthraquinone-2,6-disulphonic disodium salt (AQDS) addition obtained preferable electrochemical properties (e.g., current output, redox peak current, conductivity, electron transfer capacity) under hypersaline shock and showed superior recovery post-hypersaline stress. Partial least squares path modeling (PLS-PM) analysis indicated that the enhanced secretion of extracellular polymeric substances (EPS) and the elevated ratio of proteins to polysaccharides are likely pivotal in the formation of EB with high electrochemical activity. Meanwhile, AQDS addition constructed more complex microbial network and enriched halotolerant bacteria (e.g., Solibacillus). Higher mean function abundance of replication & repair, stress tolerance, and biofilm formation were achieved by EM addition. The results of this study present a promising approach to augment the pressure tolerance of EBs, thereby broadening the practical applications of BESs.

Evaluation of biofilm formation and carbapenem resistance in Klebsiella pneumoniae isolated from clinical samples at a rural hospital in western Uttar Pradesh

ABSTRACT Introduction: Klebsiella pneumoniae commonly causes healthcare-associated infections and shows multidrug resistance. K. pneumoniae can produce biofilm. Carbapenem resistance in K. pneumoniae is due to the production of carbapenemases mainly. This study was done to evaluate the formation of biofilm and carbapenemase resistance in K. pneumoniae isolates. Material and Methods: A total of 110 K. pneumoniae isolated from various clinical samples were taken, the antibiotic susceptibility test was done by the Kirby disk diffusion method, and biofilm detection was done by the tissue culture plate method. All the carbapenem-resistant isolates were confirmed by multiplex real-time PCR (mPCR). Those found positive for any of the carbapenemase genes were tested by the modified Hodge test (MHT), modified carbapenem inactivation method (mCIM), and ethylenediamine tetraacetic acid (EDTA)-modified carbapenem inactivation method (eCIM). Results: Out of 110 isolates, 66% (72/110) were carbapenem-resistant (suggestive of carbapenemase producers) by Kirby-Bauer disk diffusion but 58% (42/72) of Klebsiella isolates were confirmed for carbapenemase production by mPCR. Maximum number of carbapenemase gene were New Delhi metallo-β-lactamase (NDM) 52% (N = 22), 29% (N = 12) coproducers (NDM+OXA-48), and lowest in oxacillinase (OXA-48), 19% (N = 8). The overall sensitivity of MHT and mCIM+eCIM was 62% and 93%, and specificity was 88% and 97%, respectively. Our study showed that moderate biofilm producers were 51% (N = 56) K. pneumoniae isolates, strong biofilm producers 27% (N = 30), and 22% (N = 30) were weak/non-biofilm producers. We also found the correlation between biofilm formation and carbapenem-resistant K. pneumoniae (CR-KP) genes was statistically significant with a P value of 0.01*<0.05. Conclusion: Most isolates of K. pneumoniae demonstrated a wide range of antibiotic resistance and were biofilm producers. Our results indicated that the combination of mCIM with eCIM showed high sensitivity and specificity to detect CR-KP compared with MHT.

Multi-metal resistance and antibiotic susceptibility of biofilm forming bacteria isolated from Kuzhikandam Creek in Kochi, southwest part of India

The purpose of this work was to characterize and identify the bacteria isolated from highly polluted locations that could potentially be able to withstand extremely toxic heavy metals. The study area selected for bacterial isolation from sediment samples was Kuzhikandam Creek, a tributary of the Periyar River and one of the world’s most hazardous hotspots, located in Kochi’s Eloor industrial region. Heavy metal analysis of chromium, copper, and lead in sediment samples was performed, and the results showed that the concentrations were greater than permissible limits. Several metal-resistant bacteria were isolated from sediments, which, upon further screening in solid media at different concentrations of chromium, copper, and lead, found three highly resistant species (S1B1, S2B2, and S3B3), one from each site. The phenotypic and biochemical characterization of these bacteria was examined using standard procedures. The ability of these species to generate biofilm was examined using methods like the crystal violet assay (qualitative assessment) and also using the scanning electron microscope. The antibiotic susceptibility of these species was tested with six different antibiotics, which are used in various disease treatments. These isolates, sequenced using the 16S rRNA Sanger dideoxy method, have been identified as Staphylococcus arlettae, Bacillus paramycoides, and Cellulosimicrobium funkei.

Characterization of Staphylococcus aureus isolated from milk samples for their virulence, biofilm, and antimicrobial resistance

The Staphylococcus aureus (S. aureus) one of the important food borne pathogen from milk, which was investigated in this study. The isolates were screened for antimicrobial resistance, enterotoxin genes, biofilm formation, spa typing, coagulase gene polymorphism and accessory gene regulator types. The prevalence of S. aureus in milk samples was 34.4% (89/259). Methicillin resistant S. aureus (MRSA) was found at 27% (24/89) of the isolates, were classified as community acquired based on SCCmec typing. The 24.71% (22/89) isolates demonstrated multiple antimicrobial resistance (MAR) pattern. However, none of the isolates carried vancomycin and mupirocin resistance genes. The isolates were positive for sea and sed enterotoxin genes and exhibited high frequency of biofilm formation. The High-Resolution Melting and conventional spa typing revealed that the isolates had both animal and community-associated S. aureus clustered origins. Coagulase gene polymorphism and agr typing demonstrated variable genotypic patterns. The finding of this study establishes the prevalence of community associated, enterotoxigenic, biofilm forming and antimicrobial resistance among S. aureus from milk in Chennai city. This emphasizing a potential threat to public health which needs a continuous monitoring system and strategies to mitigate their spread across the food chain and achieve food safety.

Characteristics of methicillin-resistant Staphylococcus aureus isolates from bovine mastitis milk in South Korea: molecular characteristics, biofilm, virulence, and antimicrobial resistance.

This study reports on the presence and characteristics of methicillin-resistant Staphylococcus aureus strains in milk from mastitis-infected cows. To our knowledge, this is the first report of a Panton-Valentine leukocidin- and toxic shock syndrome toxin-1-positive ST22-SCCmec IV strain in South Korea.

Critical resistance to carbapenem and aminoglycosides in Pseudomonas aeruginosa: spread of blaNDM/16S methylase armA harboring isolates with intrinsic resistance mechanisms in Kerman, Iran

BackgroundCarbapenem-resistant Pseudomonas aeruginosa (CRPA) is one of the main Gram-negative bacterium causes of infections in hospital settings, and the spread of them is a significant challenge to public health.MethodsA total of 30 non-duplicate isolates of CRPA were collected. Antibacterial susceptibility of isolates to antibiotic agents, AmpC β-lactamase production, and biofilm formation were determined. Minimum biofilm inhibitory concentrations (MBIC) of isolates to cefepime (FEP), imipenem (IPM), ceftazidime (CAZ), and meropenem (MEM) were evaluated with/without cloxacillin (CLX). The carbapenemase and 16 S rRNA methylase genes were identified by PCR, and the transcription levels of oprD, ampC, and mexA genes were determined by quantitative real-time PCR (qPCR). ERIC-PCR was used to detect genetic relationships among isolates.ResultsAll isolates were multidrug resistant (MDR) and strong biofilm producers. The resistance genes including blaNDM, blaIMP, blaVIM, blaSIM, blaGES, and armA were detected in 21 (70%), 6 (20%), 3 (10%), 2 (6.6%), 1 (3.3%), and 17 (56.6%) of the isolates, respectively. CLX at 500 and 1000 µg/mL significantly reduced the level of MIC to MEM, IPM, CAZ, and FEP, also at 2000 µg/mL significantly reduced the level of MBIC to MEM, IPM, CAZ, and FEP. In all isolates, the transcription levels of oprD were significantly downregulated as well as significantly increased for ampC and mexA. ERIC-PCR typing results divided 30 isolates into four clusters A to D.ConclusionIn this study, we reported the spread of different clones of CRPA harboring co-existence of various carbapenemase genes with armA 16 S rRNA methylase for the first time in Kerman, Iran. Also, our isolates had several mechanisms of resistance to carbapenems as well as ability biofilm formation along with resistance to aminoglycosides, the further spread of which could cause serious challenges in our hospital settings. Therefore, serious monitoring is necessary to reduce their prevalence.

Co-selective effect of dissolved organic matter and chlorine on the bacterial community and their antibiotic resistance in biofilm of drinking water distribution pipes

The proliferation of pathogenic bacteria and antibiotic resistance genes (ARGs) in the biofilm of drinking water distribution pipes poses a serious threat to human health. This work adopted 15 polyethylene (PE) pipes to study the co-selective effect of dissolved organic matter (DOM) and chlorine on the bacterial community and their antibiotic resistance in biofilm. The results indicated that ozone and granular activated carbon (O3-GAC) filtration effectively removed lignins and proteins from DOM, and chlorine disinfection eliminated carbohydrate and unsaturated hydrocarbons, which both contributed to the inhibition of bacterial growth and biofilm formation. After O3-GAC and disinfection treatment, Porphyrobacter, unclassified_d_bacteria, and Sphingopyxis dominated in the biofilm bacterial community. Correspondingly, the bacterial metabolism pathways, including the phosphotransferase system, phenylalanine, tyrosine and tryptophan biosynthesis, ABC transporters, and starch and sucrose metabolism, were downregulated significantly (p < 0.05), compared to the sand filtration treatment. Under such a situation, extracellular polymeric substances (EPS) secretion was inhibited in biofilm after O3-GAC and disinfection treatment, postponing the interaction between EPS protein and pipe surface, preventing bacteria, especially pathogens, from adhering to the pipe surface to form biofilm, and restraining the spread of ARGs. This study revealed the effects of various water filtration and disinfection processes on bacterial growth, metabolism, and biofilm formation on a molecular level, and validated that the O3-GAC filtration followed by chlorine disinfection is an effective and promising pathway to control the microbial risk of drinking water.

Injectable hydrogel microsphere-bomb for MRSA-infected chronic osteomyelitis

Biofilm and bone tissue defect induced by the bacterial infection severely impede chronic osteomyelitis treatment. It is critical to break though the densely and obstinate biofilm so that the target drugs can deliver to the infected bone more effectively. Herein, an acoustically responsive multifunctional hydrogel microsphere-bomb (EMgel) was designed and prepared by microfluidic technology, which could be injected to the focus of bone infection, and blasted into the nidus deeply to destroy the bacterial biofilm matrix barrier under penetrating ultrasound, so the encapsulated natural polyphenolic EGCG and bioactive MoS2 released to repair the damaged bone. The results proved the hydrogel microsphere-bomb exhibited controlling drug release, favorable antibacterial (as high as 99 %), high biofilm resistance, fascinating antioxidation, good cytocompatibility, and osteogenic differentiation. The acoustically responsive microsphere-bomb further proved their fantastic ability to eradicate biofilm and promote bone regeneration in the Methicillin-resistant Staphylococcus aureus (MRSA) infected chronic osteomyelitis model due to the synergy effects of EGCG and bioactive MoS2. Especially, immunohistochemical staining showed lower inflammatory reaction and higher expression of OCN in EMgel group treated with ultrasound wave. This study presents a new design of hydrogel microsphere-based intelligence drug delivery for osteomyelitis treatment, which exhibit great promising potential for dealing with chronic orthopedic infections, drug delivery system and tissue engineering.

Resistance patterns, virulence determinants, and biofilm genes of multidrug-resistant Pseudomonas aeruginosa isolated from fish and fish handlers

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic bacterium that is widely distributed in aquatic environments and causes major economic losses in fish and public health hazards.This study aimed to identify the occurrence of P. aeruginosa in samples collected from fish and fish handlers, and to investigate the antimicrobial susceptibility, virulence determinants, and biofilm genes of P. aeruginosa isolates. A total of 276 samples were cross-sectionally collected from Nile tilapia (53), Golden grey mullet (52), Mediterranean horse mackerel (50), Striped red mullet (71), and fish handlers (50) at five different retail fish markets in Damietta Governorate, Egypt. Pseudomonas species (spp.) were biochemically identified in 57.9% of the total examined samples. Peudomonas aeruginosa were the most prevalent species isolated from the fish and human samples via PCR technique. Peudomonas aeruginosa isolates exhibited full resistance (100%) to tobramycin (TOB), gentamicin (CN), and colistin (CL), with a high level of susceptibility (88.5%) to imipenem (IPM) using the disk diffusion method. Most P. aeruginosa isolates (84.6%) exhibited drug resistance, with 61.5% were multidrug resistance (MDR) and 23.1% were extensive drug resistance (XDR). Most isolates had at least four virulence-associated genes (lasB, toxA, exoU, and oprL) and three biofilm genes (psIA, peIA, and lasR) by using uniplex PCR. The lasI, and rhlR Quorum Sensing (QS) genes were identified in 84.6% and 61.5% in the examined P. aeruginosa isolates, respectively. The highest mortality rate in Nile tilapia experimentally infected with P. aeruginosa isolate encoding most of virulent genes. Multivariate analyses revealed high heterogeneity among the examined isolates. This study revealed the emergence of virulent and drug resistant P. aeruginosa isolates in fish, poses high risks to consumers and food. Thus, strict hygienic measures should be considered when catching, handling, and storing fish, in addition to the routine application of antimicrobial susceptibility testing.