Biofilm Formation Ability Research Articles

Virulence genes and antibiotic susceptibility patterns of Escherichia coli isolated from nosocomial urinary tract infections in the northwest of Iran during 2022-2023: A cross-sectional study.

Urinary tract infections (UTIs) are prevalent among hospitalized patients, constituting the most frequent health-care infections. Uropathogenic Escherichia coli (UPEC) is leading causative agent of UTIs. The present study was aimed to examine the susceptibility of UPEC isolates obtained from nosocomial cases to antibiotics, as well as their biofilm formation capability and frequency of virulence genes. A total of 100 UPEC isolates were collected from nosocomial UTIs at Imam Reza Hospitals in Tabriz, Iran, spanning from April 2022 to January 2023. The antimicrobial susceptibility patterns were evaluated using the disk diffusion method, along with the detection of broad-spectrum β-lactam enzymes (ESBLs) and carbapenemases. The ability of isolates to form biofilms was assessed using the microtiter-plate method, while the PCR method was employed to identify the presence of virulence genes. The highest resistance was observed toward piperacillin (82%), followed by aztreonam and ciprofloxacin (81%), while the lowest resistance was found against piperacillin/tazobactam (12%) and meropenem (9%). ESBLs were detected in 62% of the isolates. The microtiter-plate results revealed strong, moderate, and weak biofilm formation abilities in 32%, 33%, and 24% of the isolates, respectively. The most prevalent virulence gene was fimA (74%) followed by hlyF (68%), papA (44%), papC (32%), iroN (26%), and cnf (20%). The elevated levels of resistance to multiple antimicrobial agents, coupled with the co-presence of virulence genes and biofilm formation abilities, contribute to the persistence of UPEC-related infections, particularly in hospitalized patients. These findings underscore the necessity of implementing an effective program to control nosocomial UTIs caused by UPEC in the healthcare centers.

Exploring Condition-Specific Variability in the Ureteral Stent Microbiome

(1) Background: Indwelling ureteral stents are commonly used urological devices to maintain ureteral patency, yet they have been associated with complications such as infections. Some studies have shown that bacteria adhere to and create an antimicrobial-resistant biofilm on stents. One factor that may impact biofilm formation is the original condition informing stent placement, such as kidney stones and renal allografts. Both kidney stones and renal allografts are independently associated with infection, yet the differential stent microbiomes of these populations remain poorly characterized. Our objective was to characterize these microbiomes in order to inform urological health practice and help prevent ureteral stent-associated infections. (2) Methods: Stents were collected from kidney stone and renal transplant recipients undergoing routine cystoscopic stent removal. Microbial DNA was extracted from stents and analyzed using 16S Next Generation Sequencing. Descriptive statistics, alpha diversity, and beta diversity methods were used for statistical analysis. (3) Results: The microbiome of ureteral stents in kidney stone and transplant patients is composed of unique species, each with different biofilm-forming abilities. (4) Conclusions: Our findings demonstrate that the microbiome of stents differs based on preceding condition. It is important to conduct future studies that explore this microbiome further to understand what type of stent-associated infection someone may develop based on their initial condition.

Draft genomes of the bile duct microbiome strains Klebsiella pneumoniae and Enterococcus lactis isolated from bilioenteric drainages with biofilm-forming abilities.

We describe the genetic properties of two strains isolated from the elusive bile duct microbiome from solid organ transplant patients. Bacterial strains Enterococcus lactis (MS-STENT-08-E-001) and Klebsiella pneumoniae (MS-STENT-01-M-001) were isolated from the biofilms of bile duct catheters.

Synergistic effects of bacteriophage cocktail and antibiotics combinations against extensively drug-resistant Acinetobacter baumannii

BackgroundThe extensively drug-resistant (XDR) strains of Acinetobacter baumannii have become a major cause of nosocomial infections, increasing morbidity and mortality worldwide. Many different treatments, including phage therapy, are attractive ways to overcome the challenges of antibiotic resistance.MethodsThis study investigates the biofilm formation ability of 30 XDR A. baumannii isolates and the efficacy of a cocktail of four tempetate bacteriophages (SA1, Eve, Ftm, and Gln) and different antibiotics (ampicillin/sulbactam, meropenem, and colistin) in inhibiting and degrading the biofilms of these strains.ResultsThe majority (83.3%) of the strains exhibited strong biofilm formation. The bacteriophage cocktail showed varying degrees of effectiveness against A. baumannii biofilms, with higher concentrations generally leading to more significant inhibition and degradation rates. The antibiotics-bacteriophage cocktail combinations also enhanced the inhibition and degradation of biofilms.ConclusionThe findings suggested that the bacteriophage cocktail is an effective tool in combating A. baumannii biofilms, with its efficacy depending on the concentration. Combining antibiotics with the bacteriophage cocktail improved the inhibition and removal of biofilms, indicating a promising strategy for managing A. baumannii infections. These results contribute to our understanding of biofilm dynamics and the potential of bacteriophage cocktails as a novel therapeutic approach to combat antibiotic-resistant bacteria.

Identification and characterization of LuxR solo homolog PplR in pathogenic Pseudomonas plecoglossicida NB2011

Pseudomonas plecoglossicida is a causative agent of visceral granulomas in large yellow croaker (Larimichthys crocea). Quorum sensing (QS) is widely involved in imparting virulence to pathogenic bacteria; however, it has not been studied in P. plecoglossicida. In this study, we annotated a LuxR family transcriptional regulator in P. plecoglossicida NB2011 and designated as PplR. We aligned the protein sequence by BlastP and Clustal X2, monitored the N-acyl-homoserine lactone (AHL) signal production through cross-feeding bioassay and HC-MS/MS; investigated exogenous AHL signal binding by recombinant expression and thin layer chromatography; constructed a deletion mutant of the target gene by method of double homologous recombination; sequenced the transcript RNA and analyzed the data; additionally, characterized phenotypes of wild type and mutant strain. The LuxR homolog PplR was found to share high similarity with PpoR—the LuxR solo of Pseudomonas putida—without a cognate LuxI. The wild-type strain did not produce any AHL signals and the recombinant LuxR protein was found to bind C6-L-homoserine lactone (C6-HSL), C8-HSL, 3-oxo-C10-HSL, and 3-oxo-C12-HSL. RNA-seq analysis indicated 84 differentially expressed genes—5 upregulated and 79 downregulated—mainly enriched in gene ontology terms, such as flagella-dependent motility, integral component of membrane, DNA binding and transcription, and metal ion binding, suggesting that PplR is a master transcription regulator. The mutant strain showed attenuated biofilm-forming ability and stress resistance, and the data support a role for PplR in the regulation of these traits in P. plecoglossicida NB2011 independent of the presence of AHL signals. This is the first study to provide QS-related information on P. plecoglossicida.

Antisalmonella Activities of Essential Oil of Psidium guajava Leaves: An In vitro and In vivo Study

Background: Life-threatening typhoidal salmonella infection is becoming more difficult to treat due to rising resistance to commonly used antibiotics. This resistance is aided by the biofilm-forming ability of the bacteria. This study investigated the in vitro and in vivo antisalmonella activities of essential oil of Psidium guajava leaves. Methods: Essential oil was extracted from the leaves of Psidium guajava with solvents. The sensitivity of Salmonella Typhi to the essential oil of Psidium guajava (EOPG) was assessed via the agar well diffusion method. Minimum inhibitory, bactericidal and biofilm-inhibitory concentrations (MIC, MBC and MBIC respectively) were measured using micro-broth dilution and crystal violet assay respectively. The effect of EOPG on the bacterial cell membrane was evaluated by measuring the efflux of potassium ions, inorganic phosphate and pyruvic acid. Bioautography and GC-MS were employed to identify the constituents of the active fraction of the EOPG. Effects of EOPG on the level of bacteremia and bone marrow infection in male Wistar rats were determined by plating caudal blood on salmonella-shigella agar. Selected serum cytokines were measured using ELISA kits. Effect on hepatic DNA was measured by comet assay while histological evaluation of the liver of infected rats was done by Hematoxylin and Eosin staining. Results: Essential oil of Psidium guajava leaves (EOPG) inhibited the growth of S. Typhi with a zone of inhibition of 13±0.2mm compared to 31±0.3mm recorded by ciprofloxacin. Minimum inhibitory, minimum bactericidal and minimum biofilm inhibitory concentrations were 25, >50 and 12.5 mg/mL respectively. Exposure to EOPG caused efflux of potassium ions, inorganic phosphate and pyruvic acid in S. Typhi. Oral administration of 50 mg/mL of EOPG reduced bacterial load in the blood and bone marrow of S. Typhi-infected rats significantly (P<0.05) when compared to untreated animals. Infection-induced reduction of serum IL-1β and rise in serum IFN-γ were ameliorated considerably in treated animals. The hepatic DNA of infected animals were protected from breakage in the group administered 50 mg/mL/day of EOPG compared with untreated, ciprofloxacin-treated as well as those administered 100 mg/mL/day of EOPG. Conclusion: EOPG has both in vitro and in vivo anti-salmonella activities, this could be exploited in developing new drug leads for treatment of typhoid fever

2,4-Di-Tert-Butylphenol of Streptomyces luridiscabiei MMS-10 Inhibits Biofilm Forming Cariogenic Streptococcus mutans ULSP-2.

Dental caries is a common chronic infectious disease of the oral cavity that affects the overall oral health of the individual. Cariogenic bacteria have long been recognized for their role in developing chronic dental infections. Drug-resistant bacteria represent a global challenge to effective pathogen control in caries. The present study aimed to isolate and identify soil actinomycetes for their antibacterial and anti-biofilm activities against antibiotic-resistant and biofilm-forming cariogenic bacteria. Thirteen caries bacteria isolated from infected tooth samples were evaluated for antibiotic resistance and biofilm formation. The isolate ULSP-2 showed the highest antibiotic resistance score (0.714) and was found to be a strong biofilm producer when tested by congo red agar and microtiter plate assays. The bacterium was identified as Streptococcus mutans based on morphological, biochemical, and molecular characterization. The effect of ethyl acetate extracts from 20 soil actinomycetes on the growth and biofilm formation ability of S. mutans was evaluated. The MMS-10 extract strongly inhibited growth (18.5 ± 0.5mm) and biofilm formation (56.46 ± 0.32%) of S. mutans at 100µg/mL. The isolate MMS-10 was identified at the molecular level as Streptomyces luridiscabiei. Based on FTIR, NMR, and GC-MS analysis, the purified MMS-10 extract was characterized and identified as 2,4-Di-tert-butylphenol. The metabolite's physiological, physicochemical, and pharmacokinetic properties were analyzed using the Swiss ADME web server and found to satisfy the criteria of drug-likenessof a molecule. The study revealed the significance of soil actinomycetes in controlling growth and biofilm formation in cariogenic S. mutans.

Unveiling the Potential of CuO and Cu2O Nanoparticles against Novel Copper-Resistant Pseudomonas Strains: An In-Depth Comparison.

Four novel Pseudomonas strains with record resistance to copper (Cu2+) previously isolated from ecologically diverse samples (P. lactis UKR1, P. panacis UKR2, P. veronii UKR3, and P. veronii UKR4) were tested against sonochemically synthesised copper-oxide (I) (Cu2O) and copper-oxide (II) (CuO) nanoparticles (NPs). Nanomaterials characterisation by X-ray diffractometry (XRD), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR), and High-Resolution Transmission Electron Microscopy (HRTEM) confirmed the synthesis of CuO and Cu2O NPs. CuO NPs exhibited better performance in inhibiting bacterial growth due to their heightened capacity to induce oxidative stress. The greater stability and geometrical shape of CuO NPs were disclosed as important features associated with bacterial cell toxicity. SEM and TEM images confirmed that both NPs caused membrane disruption, altered cell morphology, and pronounced membrane vesiculation, a distinctive feature of bacteria dealing with stressor factors. Finally, Cu2O and CuO NPs effectively decreased the biofilm-forming ability of the Cu2+-resistant UKR strains as well as degraded pre-established biofilm, matching NPs' antimicrobial performance. Despite the similarities in the mechanisms of action revealed by both NPs, distinctive behaviours were also detected for the different species of wild-type Pseudomonas analysed. In summary, these findings underscore the efficacy of nanotechnology-driven strategies for combating metal tolerance in bacteria.

Staphylococcus pseudintermedius ST2660 isolated from a cat has strong biofilm-forming ability and increases biofilm formation at cat’s normal body temperature

Staphylococcus pseudintermedius has been isolated from dogs, cats, and horses and is also known as an emergent zoonotic agent. We administered orbifloxacin, a fluoroquinolone, to treat bacterial infections of cutaneous wounds caused by excessive grooming of the skin in contact with the subcutaneous port of the subcutaneous ureteral bypass (SUB) system in a cat. However, after 80 days of treatment, a severe abscess was observed in the wound and fluoroquinolone-resistant S. pseudintermedius was isolated from the abscess. The isolate was identified as a novel sequence type (ST) 2660 and contained genes for leukocidins (lukS and lukF), exfoliative toxin (siet), and biofilm regulation (icaA and icaD). The isolate was resistant to macrolide, lincosamide, fluoroquinolone, and tetracycline classes. In addition, the isolate had strong biofilm-forming ability which significantly increased with culturing at 39 °C compared with that at 37 °C, suggesting that the isolate prefers a cats’ body temperature as the optimal biofilm growth condition. Notably, the biofilms were increased in the presence of doxycycline with culturing at 39 °C. This study is the first report in Japan on the new sequence type of S. pseudintermedius isolated from a companion animal and clarifies the distinctive virulence of S. pseudintermedius.

Biofilm Prevention and Removal in Non-Target Pseudomonas Strain by Siphovirus-like Coliphage.

Background/Objectives. Bacteriophages have gained significant interest as a potential solution to combat harmful bacteria, especially in the fight against antimicrobial resistance. With the rise in drug-resistant microorganisms, the medical community is increasingly exploring new alternatives to traditional antibiotics, and bacteriophages offer several advantages in this regard. However, phage applications still face some challenges, such as host specificity. Methods. In this study, a somatic Siphovirus-like coliphage (SOM7) was tested for inhibiting the biofilm-forming capacity of the non-target strain Pseudomonas aeruginosa (ATTC 10145). The phage-sensitive strain E. coli WG5 was used as a control. The selected microorganisms were first tested for growth in the presence of SOM7 at three different concentrations (105, 107, and 109 PFU/mL). Results. As expected, the phage-sensitive E. coli WG5 was fully inhibited by the coliphage, and no phage-related affection on the growth rate was observed for the SOM7-resistant P. aeruginosa. More notably, increasing concentrations of SOM7 significantly reduced both the biofilm-forming capacity and the amount of pre-established bacterial biofilm of the phage-insensitive P. aeruginosa (24.9% and 38.8% reduction in the biofilm-forming ability, and 18.8% and 28.0% biofilm degradation for 107 PFU/mL and 109 PFU/mL SOM7, respectively; p < 0.05). These results were supported by transmission electron microscopy (TEM) imaging, providing unprecedent evidence for the interaction of the somatic coliphage with the non-host strain. Conclusions. Although more studies in other biofilm models are necessary, our results show for the very first time that bacteriophages could potentially be used as an alternative to achieve desired anti-biofilm and biofilm-degrading activity in non-host bacterial strains.

Molecular epidemiological analysis of blaNDM-5-producing Klebsiella pneumoniae ST2407-K25 causing infection outbreaks in pediatric patients based on whole genome sequencing

BackgroundPediatric patients are vulnerable to the threat of carbapenem-resistant Klebsiella pneumoniae (CRKP) due to their limited immunity and few available antibiotics. Especially when these pathogens exhibit hypervirulent phenotypes, they are often associated with poor clinical outcomes.MethodsIn this study, we investigated a CRKP outbreak in pediatric patients from 2019 to 2021 in a teaching hospital in China based on whole genome sequencing. We sequenced twenty-nine CRKP isolates isolated from unduplicated pediatric patients to understand their genetic relationships, virulence factors, resistance mechanisms, and transmission trajectories. Conjugation experiments were performed to evaluate the horizontal transfer ability of carbapenem resistance determinants in twenty-nine CRKP isolates. We then characterized these isolates for biofilm formation ability and serum resistance. Genetic relatedness, comparison of plasmids, and chromosomal locus variation of CRKP isolates were analyzed by bioinformatics.ResultsAll the isolates were carbapenemase-producers harbouring blaNDM−5. Among them, twenty-eight isolates belonged to the ST2407 group, with the consistent capsular serotype K25. The virulence-related factors: ureA, fim, ybtA, irp1/irp2, and mrkA were prevalent in these isolates. Additionally, most CRKP isolates showed moderately adherent biofilm formation. Although the ST2407 clonal group did not exhibit serum resistance, the heterogeneous level of serum resistance was related to the disruption of oqxR. Conjugation and WGS revealed that the blaNDM−5 carried by the twenty-eight CRKP ST2407 isolates was located on nonconjugative IncX3 plasmids associated with deleting the T4SS-encoding genes. Clonal transmission of CRKP ST2407 in pediatric patients was suggested by the phylogenetic tree.ConclusionsOur study provides evidence of the clonal spread of blaNDM−5-producing K. pneumoniae in pediatric patients and the necessity for the T4SS system for horizontal transfer of the IncX3 plasmid carrying blaNDM−5. Additionally, the disruption of oqxR may have affected the serum resistance of CRKP. The results of this study emphasize the importance of continuously monitoring for CRKP infection in pediatric patients to prevent recurrent infections.

ISOLATION AND CHARACTERIZATION OF BIOFILM FORMING ESCHERICHIA COLI FROM CHICKEN MEAT SAMPLES

The present study was aimed to investigate the presence of Escherichia coli (E.coli) in raw chicken meat samples collected from retail shops, as well as the biofilm-forming ability of field isolates, and to characterize different adhesion genes. Out of 20 chicken meat samples, 17 (85%) were positive for E. coli. Fifteen E. coli strains were characterized by PCR using the 16S rDNA primers and all the isolates were positive which confirmed that all the isolates were E.coli. Out of the 15 confirmed E.coli field isolates which were subjected to biofilm-forming assay, 46% of them were found to be strong biofilm producers. While all the isolates were screened for the presence of adhesion genes viz. luxS, csgA, fimH, fimA, and papC, the adhesion gene luxS was detected in all the strains (100%). The other adhesion genes csgA, fimH and fimA were detected in 93%, 93%, and 73% of the isolates, respectively. The E. coli field isolates were screened blaTEM gene was detected in only four strains, which was categorized under strong biofilm producers. This study demonstrated the presence of biofilm forming E. coli in the raw chicken meat samples as contaminants, causing spoilage and potentially posing risk to consumer’s health and safety.

Screening and functional characterization of isocitrate lyase AceA in the biofilm formation of Vibrio alginolyticus.

Biofilm is a well-known sessile lifestyle for bacterial pathogens, but a little is known about the mechanism on biofilm formation in Vibrio alginolyticus. In this study, we screened V. alginolyticus strains with strong biofilm formation ability from coastal seawater. The antibiotic resistance of the biofilm cells (BFs) was higher than that of the planktonic cells (PTs). To study the genes and pathways involved in biofilm formation, we performed transcriptome analysis of the BFs and PTs of V. alginolyticus R9. A total of 685 differentially expressed genes (DEGs) were upregulated, and 517 DEGs were downregulated in the BFs. The upregulated DEGs were significantly enriched in several pathways including glyoxylate and dicarboxylate metabolism, while the downregulated genes were significantly enriched in the flagellar assembly pathways. The key gene involved in glyoxylate shunt, aceA, was cloned, and ΔaceA mutant was constructed to determine the function of AceA in carbon source utilization, biofilm formation, and virulence. Real-time reverse transcription PCR showed that the expression of aceA was higher at the mature stage but lower at the disperse stage of biofilm formation, and the expression of the flagellar related genes was upregulated in ΔaceA. This is the first study to illustrate the global gene expression profile during the biofilm formation of V. alginolyticus, and isocitrate lyase AceA, the key enzyme involved in glyoxylate shunt, was shown to maintain biofilms accompanied by downregulation of flagellation but promoted dispersal of BFs at the late stage.IMPORTANCEBiofilms pose serious public problems, not only protecting the cells in it from environmental hazard but also affecting the composition and abundance of bacteria, algae, fungi, and protozoa. The important opportunistic pathogen Vibrio alginolyticus is extremely ubiquitously present in seawater, and it also exhibited a strong ability to form biofilm; thus, investigation on the biofilm formation of V. alginolyticus at molecular level is fundamental for the deeper exploration of the environmental concerns arose by biofilm. In this study, transcriptome analysis of biofilm cells (BFs) and planktonic cells (PTs) from V. alginolyticus was performed and AceA was screened to play an important role in biofilm formation. AceA was shown to maintain biofilms accompanied by downregulation of flagellation but promoted dispersal of BFs at the disperse stage. This method was helpful to further understand the ability and mechanism of V. alginolyticus biofilm formation and provide clues for prevention of V. alginolyticus infection.

Repurposing simvastatin for treatment of Klebsiella pneumoniae infections: in vitro and in vivo study

Simvastatin had minimum inhibitory concentrations of 32 to 128 µg/mL against Klebsiella pneumoniae isolates and hindered the biofilm-formation ability of 58.54% of the isolates. It considerably diminished the bacterial cell counts in the biofilms as revealed by scanning electron microscope. Also, qRT-PCR revealed a downregulation of the biofilm genes (bcsA, wza, and luxS) by simvastatin in 48.78% of the isolates. Moreover, simvastatin has significantly improved the survival of mice and decreased the burden of bacteria in the infected lungs. Also, the histological architecture was substantially improved in the simvastatin-treated group, as the alveolar sacs and bronchioles appeared normal with minimal collagen fiber deposition. The immunohistochemical studies exposed that the TNF-α, NF-kβ, and COX-2 immunostaining considerably declined in the simvastatin-treated group. Furthermore, ELISA exposed that both IL-1β and IL-6 were considerably diminished in the lungs of the simvastatin-treated group.

Wastewater-based surveillance of Vibrio cholerae: Molecular insights on biofilm regulatory diguanylate cyclases, virulence factors and antibiotic resistance patterns

Vibrio cholerae is an inherent inhabitant of aquatic ecosystems. The Indian state of West Bengal, especially the Gangetic delta region is the highest cholera affected region and is considered as the hub of Asiatic cholera. V. cholerae were isolated from publicly accessible wastewater of Midnapore, West Bengal, India. Serotyping determined all isolates to be of non-O1/non-O139 serogroups. Moderate biofilm-forming abilities were noticed in most of the isolates (74.7 %) while, high biofilm formation was recorded for only 6.3 % isolates and 19 % of isolates exhibited low/non-biofilm-forming abilities. PCR-based screening of crucial diguanylate cyclases (DGCs) involved in cyclic-di-GMP-mediated biofilm signaling was performed. cdgH and cdgM were the most abundant DGCs among 93.7 % and 91.5 % of isolates, respectively. Other important DGCs, i.e., cdgK, cdgA, cdgL, and vpvC were present in 84 %, 75.5 %, 72 % and 68 % of isolates, respectively. Besides, the non-O1/non-O139 isolates were screened for the occurrence of virulence factor encoding genes. Moreover, among these non-O1/non-O139 isolates, two strains (3.17 %) harbored both ctxA and ctxB genes, which encode the cholera toxin associated with epidemic cholera. ompU was the most prevalent virulence factor, present in 24.8 % of isolates. Other virulence factors like, zot and st were found in 4.7 % and 9.5 % of isolates. Genes encoding tcp and ace were found to be PCR-negative for the isolates. Additionally, crucial virulence factor regulators, toxT, toxR and hapR were found to be PCR-positive in all the isolates. Antibiotic resistance patterns displayed further vulnerabilities with decreased sensitivity towards commonly used antibiotics with multiple antibiotic resistance index ranging between 0.37 and 0.62. The presence of cholera toxin-encoding multi-drug resistant (MDR) V. cholerae strains in environmental settings is alarming. High occurrence of DGCs are considered to encourage further investigations to use them as alternative therapeutic targets against MDR cholera pathogen due to their unique presence in bacterial systems.

Assessment of prevalence, antibiotic resistance, and virulence profiles of biofilm-forming Enterococcus faecalis isolated from raw seafood in Bangladesh

Enterococcus faecalis are often resistant to different classes of antibiotics, harbor virulence determinants, and produce biofilm. The presence of E. faecalis in raw seafood exhibits serious public health significance. This study aimed to identify antibiotic resistance patterns and virulence factors in biofilm-forming E. faecalis strains extracted from seafood in Bangladesh. A total of 150 samples of raw seafood, comprising 50 shrimps, 25 crabs, and 75 fish, were collected and subjected to culturing, biochemical, and PCR assays to detect E. faecalis. The biofilm-forming abilities of the isolates were determined by Congo Red agar (CRA) plate and Crystal Violet Micro-titer Plate (CVMP) tests. Antibiotic resistance profiles were evaluated using the disk diffusion method. Virulence genes of the isolates were detected by PCR assay. The occurrence of E. faecalis was 29.3 % (44/150), which was higher in crabs and fish (36 %) than in shrimps (16 %). In CRA and CVMP tests, biofilm-forming abilities were observed in 88.64 % of the isolates, whereas 11 (25 %) and 28 (63.6 %) were strong- and intermediate-biofilm formers, respectively. All the isolates contained at least two virulence genes, including pil and ace (97.7 %), sprE (95.5 %), gelE (90.9 %), fsrB (79.6 %), agg (70.5 %), fsrA (68.2 %), and fsrC (61.4 %). All the isolates were phenotypically resistant to penicillin, followed by ampicillin and rifampicin (86.4 %), erythromycin (13.7 %), and tetracycline, vancomycin, norfloxacin, and linezolid (2.3 %). Resistant gene blaTEM was found in 61.4 % of the isolates. Moreover, the study found that E. faecalis strains with strong biofilm-forming capabilities had significantly higher levels of virulence genes and antibiotic resistance (p < 0.05) compared to those with intermediate and/or no biofilm-forming abilities. To the best of our knowledge, this research represents the first instance in Bangladesh of assessing antibiotic resistance and identifying virulence genes in biofilm-forming E. faecalis strains isolated from seafood samples. Our study revealed that seafood is a carrier of antibiotic-resistant, virulent, and biofilm-forming E. faecalis, demonstrating a potential public health threat.

Emergence of extensively drug-resistant hypervirulent Acinetobacter baumannii isolated from patients with bacteremia: bacterial phenotype and virulence analysis

ObjectivesIndividuals infected with extensively drug-resistant (XDR) Acinetobacter baumannii are difficult to cure and have a high mortality rate. In this study, we compared the genomic and phenotypic differences between XDR and non-multidrug-resistant (MDR) A. baumannii and further characterized hypervirulent XDR A. baumannii. MethodsA total of 1,403 Acinetobacter isolates were collected from patients with bacteremia between 1997 and 2015. Antimicrobial susceptibility test was performed to categorize isolates into non-MDR, MDR, and XDR groups. The presence of selected virulence-associated genes was determined by PCR. Bacterial phenotypes, including iron acquisition, biofilm formation, capsule production, and virulence to larvae and mice, were determined. ResultsMultilocus sequence types revealed the high prevalence of ST2 (81.6%) and ST129 (18.4%) among 49 XDR isolates and sequence types of 18 non-MDR isolates were more diverse. Virulence-associated phenotypic assays showed that XDR isolates had higher iron acquisition ability and capsule production and higher virulence to Galleria mellonella larvae. However, their biofilm formation ability was lower compared to that of non-MDR isolates. XDR isolates contained more virulence genes, tonB, hemO, abaI, and ptk, while non-MDR isolates contained more pld and ompA. Twenty-one XDR isolates caused <20% larvae survival rate after 7 days post-infection were defined as hypervirulent XDR isolates. Among them, isolates 1677 (ST129) and 929-1 (ST2) also caused the death of all infected mice within two days. ConclusionSome subpopulations of highly drug-resistant ST2 also exhibit high virulence. Therefore, it is of utmost importance to continue monitoring the spread of hypervirulent XDR A. baumannii isolates.

Emergence and spread of resistant and biofilm-forming Acinetobacter baumannii in critically ill COVID-19 patients

The COVID-19 pandemic has raised concerns beyond the viral infection itself. Bacterial co-infections, particularly those involving Acinetobacter baumannii, have become a significant worry in critically ill COVID-19 patients. A. baumannii is an opportunistic pathogen that can cause nosocomial infections, especially in patients with compromised immune systems. This study investigated 36 A. baumannii isolates obtained from COVID-19 patients during a concurrent outbreak. The isolates were collected over two years through routine medical requests sent to the Clinical Microbiology laboratory. Identification of the strains was confirmed through biochemical tests, the Phoenix BD® Automated Microbiology System, and MALDI-TOF Mass Spectrometry. The study assessed the antimicrobial sensitivity of the isolates, with a specific focus on resistance to the beta-lactam group as well as aminoglycosides. The presence of specific antibiotic resistance genes (blaOXA-23, -24, -51 and -58, blaKPC, blaSHV, blaIMP, blaVIM, aac(6')-Ib, ant(3'')-Ia, and aph(3')-Ia) was investigated using PCR and Sanger DNA sequencing. Biofilm-forming capabilities of the isolates were also evaluated. The findings revealed diverse resistance profiles, with a high prevalence of resistant strains, including resistance to carbapenems. Genetic analysis suggested potential clonal spread of certain strains within the hospital setting. Moreover, a significant proportion of the isolates demonstrated strong biofilm-forming abilities, which can enhance persistence and antibiotic resistance. In conclusion, this study highlights the need for vigilant monitoring and targeted interventions to address bacterial co-infections in COVID-19 patients. The diversity in resistance patterns, potential clonal spread, and robust biofilm-forming abilities among A. baumannii isolates underscore the importance of addressing this issue to better manage and treat critically ill COVID-19 patients.

Identification and comparison of protein composition of biofilms in response to EGCG from Enterococcus faecalis and Staphylococcus lugdunensis, which showed opposite patterns in biofilm-forming abilities.

Bacterial biofilm is resistant to conventional antibiotic treatments, leading to complications associated with many infection-related human diseases. Epigallocatechin Gallate (EGCG), a phenolic catechin enriched in green tea, is recognized for its anti-bacterial and anti-biofilm activities. In this study, we examined the protein components of the biofilms formed in the absence or presence of EGCG using Enterococcus faecalis and Staphylococcus lugdunensis, which had shown opposing patterns in biofilm formation. A clustering heatmap revealed that the two microorganisms expressed the different protein sets in response to EGCG. Proteins that were noticeably upregulated included those associated with stress responsiveness and gluconeogenesis in E. faecalis, and gene modification in S. lugdunensis. Conversely, downregulated proteins were related to tRNA-modifying enzyme activity in E. faecalis, and anabolic metabolism in S. lugdunensis. Among the proteins identified only in EGCG-responsive biofilms, enzymes involved in de novo purine biosynthesis were enriched in E. faecalis, while proteins likely to cause DNA instability and pathogenicity changes were abundantly present in S. lugdunensis. The classification based on gene ontology (GO) terms by microorganism exhibited that metabolic process or catabolic activity was at the top rank in E. faecalis with more than 33 proteins, and in S. lugdunensis, localization or transport was highly ranked with 4 proteins. These results support the hypothesis that EGCG might cause different cellular programs in each microorganism. Finally, comparison of the proteomes between two groups that form biofilms to similar extents discovered that 2 proteins were commonly found in the weak biofilm-forming groups (E. faecalis and EGCG-responding S. lugudunensis), whereas 9 proteins were common among the strong biofilm-forming groups (S. lugdunensis and EGCG-responding E. faecalis). It was suggested that these proteins could serve as potential indicators to detect the presence and predict the extent of biofilm formation by multiple microorganisms. Taken all together, proteomics data and analyses performed in this study provided useful and new information on the proteins embedded in the biofilms formed at the specific conditions, which can aid in diagnosis and the development of tailored treatment strategies.

Hypervirulent Carbapenem-Susceptible Klebsiella pneumoniae ST412/K57 with Strong Biofilm Formation: association with gas gangrene and sepsis.

Hypervirulent Klebsiella pneumoniae (hvKp) poses a serious public health threat. Gas gangrene caused by hvKp was rarely reported, potentially resulting in a poor prognosis. In this study, we described the case of a hospitalized patient with gas gangrene and sepsis by hvKP. The carbapenem-susceptible hypervirulent Klebsiella pneumoniae (CS-hvKp) strains KPLSN and KPLSX were isolated from the knee joint pus and blood specimens of the patient for further investigations. Whole genome sequencing revealed that KPLSN and KPLSX were highly homologous (SNPs<10) and belonging to ST412/K57. The minimum inhibitory concentration and minimum bactericidal concentration under biofilm values of meropenem in KPLSN and KPLSX were significantly higher than planktonic state (>128 mg/L versus 0.25 mg/L, P<0.0001). These two strains had high biofilm formation ability, and fluorescence staining experiments results showed that they were not easily killed by meropenem in the biofilm state. KPLSN and KPLSX showed high capsular production and were confirmed with high virulence through experiments with the Galleria mellonella and BALB/c mice abdominal infection model. The persistent symptoms may be due to enhanced biofilm and capsule formation. Global ST412 strains phylogenetic analysis showed their evolution towards higher virulence and resistance. It emphasizes the critical need for judicious antibiotic use and novel therapeutic approaches to combat special infections caused by these pathogens.