Saeed A. Khan

Staphylococcus epidermidis biofilms are inherently tolerant to antimicrobial treatment, contributing to the persistence and recurrence of biofilm-associated infections. In this study, we examined the phenotypic and proteomic responses of S. epidermidis ATCC 35984 biofilms following exposure to subinhibitory concentrations (1/8× MIC) of levofloxacin, rifampicin, and their combination. Phenotypic analyses revealed clear differences among the treatments. Rifampicin significantly reduced the number of viable biofilm-associated cells, whereas levofloxacin alone had little effect. The combination of rifampicin and levofloxacin produced the most pronounced antibiofilm activity, significantly reducing both biofilm biomass and viability. These findings were supported by confocal laser scanning microscopy, which showed the greatest loss of viable cells and biofilm integrity in the combination-treated biofilms. Label-free quantitative proteomic analysis demonstrated distinct treatment-specific adaptive responses. Rifampicin, either alone or in combination with levofloxacin, triggered broadly increased abundance of proteins involved in translation, energy production, amino acid and nucleotide metabolism, ATP-binding cassette transport, and quorum sensing. In contrast, levofloxacin predominantly suppressed proteins associated with central metabolism, transcription, translation, and biosynthetic pathways. Hierarchical clustering further showed that the proteomic profile of the combination treatment closely resembled that of rifampicin alone, suggesting that rifampicin was the dominant driver of proteomic remodeling. Expression pattern network analysis identified a conserved stress-response signature comprising Fhs, YdaG, PerR, and YcsE across all treatment conditions. Notably, the multidrug efflux pumps NorA and NorC showed decreased protein abundance across all treatment conditions, indicating a potential reduction in efflux-mediated resistance under sub-MIC antibiotic exposure. Taken together, these findings demonstrate that rifampicin retains substantial antibiofilm activity at subinhibitory concentrations and elicits extensive metabolic adaptation, whereas levofloxacin primarily induces a metabolically repressed state. The enhanced activity of the rifampicin–levofloxacin combination appears to arise from complementary effects on biofilm viability and cellular physiology. This study provides new insights into the adaptive responses of S. epidermidis biofilms to sublethal antibiotic stress and highlights the potential of rifampicin-based combination therapies for the management of biofilm-associated infections.

Urinary tract infections (UTIs) remain a major global health concern, with rising antimicrobial resistance prompting the search for alternative therapies. Selenium nanoparticles (Se NPs) are promising antimicrobial agents due to their unique physicochemical properties and ability to disrupt bacterial physiology. This study evaluated the antibacterial efficacy of Se NPs against four uropathogens and conducted comparative proteomic analyses to elucidate stress responses. Enumeration assays showed that Se NPs effectively inhibited bacterial growth, with Pseudomonas aeruginosa being the most susceptible and Proteus mirabilis the most resistant. Microscopy revealed Se NP-induced membrane rupture and cellular deformation across all species. Proteomic and bioinformatic analyses showed more pronounced protein regulation in P. mirabilis than in P. aeruginosa. Cluster of Orthologous Groups (COG) analysis revealed both shared and species-specific responses, while Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated activation of key stress pathways. Virulence-associated proteins were modulated in both species, with P. mirabilis uniquely upregulating stress survival and exotoxin-related proteins. Both regulated efflux pumps, suggesting active transport mitigates Se NP toxicity. P. aeruginosa showed mercury resistance, while P. mirabilis expressed tellurite resistance proteins. These findings highlight distinct yet overlapping strategies and support the potential of Se NPs in novel antimicrobial development.

We report here the complete genome sequence of cephalosporin (cefazolin and cephalotin) and tetracycline-resistant Citrobacter freundii strain CF51, isolated from a patient with a urinary tract infection. The genome comprises a 5.0-Mb chromosome, along with an 87.2-kb plasmid and a 23.9-kb plasmid.

Our whole-genome sequencing analysis of sixteen uropathogenic E. coli isolates revealed a concerning picture of multidrug resistance and potentially virulent bacteria. All isolates belonged to four distinct clonal groups, with the highly prevalent ST131 lineage being associated with extensive antibiotic resistance and virulence factors. Notably, all isolates exhibited multidrug resistance, with some resistant to as many as 12 antibiotics. Fluoroquinolone resistance stemmed primarily from efflux pumps and mutations in gyrase and topoisomerase genes. Additionally, we identified genes encoding resistance to extended-spectrum cephalosporins, trimethoprim/sulfamethoxazole, and various heavy metals. The presence of diverse plasmids and phages suggests the potential for horizontal gene transfer and the dissemination of virulence factors. All isolates harbored genomic islands containing virulence factors associated with adhesion, biofilm formation, and invasion. Genes essential for iron acquisition, flagella biosynthesis, secretion systems, and toxin production were also prevalent. Adding further complexity to understanding the isolates’ genetic makeup, we identified CRISPR-Cas systems. This study underscores the need for continued genomic surveillance in understanding the pathogenic mechanisms and resistance profiles of uropathogenic E. coli to aid in developing targeted therapeutic strategies.

Campylobacter jejuni is the foodborne pathogen causing most gastrointestinal infections. Understanding its ability to form biofilms is crucial for devising effective control strategies in food processing environments. In this study, we investigated the growth dynamics and biofilm formation of C. jejuni NCTC 11168 in various culture media, including chicken juice (CJ), brain heart infusion (BHI), and Mueller Hinton (MH) broth. Our results demonstrated that C. jejuni exhibited a higher growth rate and enhanced biofilm formation in CJ and in 1:1 mixtures of CJ with BHI or MH broth compared to these measures in BHI or MH broth alone. Electron microscopy unveiled distinct morphological attributes of late-stage biofilm cells in CJ, including the presence of elongated spiral-shaped cells, thinner stretched structures compared to regular cells, and extended thread-like structures within the biofilms. Proteomic analysis identified significant alterations in protein expression profiles in C. jejuni biofilms, with a predominance of downregulated proteins associated with vital functions like metabolism, energy production, and amino acid and protein biosynthesis. Additionally, a significant proportion of proteins linked to biofilm formation, virulence, and iron uptake were suppressed. This shift toward a predominantly coccoid morphology echoed the reduced energy demands of these biofilm communities. Our study unlocks valuable insights into C. jejuni’s biofilm in CJ, demonstrating its adaptation and survival.

We analyzed antimicrobial resistance and virulence traits in multidrug-resistant (MDR) E. coli isolates obtained from imported shrimp using whole-genome sequences (WGSs). Antibiotic resistance profiles were determined phenotypically. WGSs identified key characteristics, including their multilocus sequence type (MLST), serotype, virulence factors, antibiotic resistance genes, and mobile elements. Most of the isolates exhibited resistance to gentamicin, streptomycin, ampicillin, chloramphenicol, nalidixic acid, ciprofloxacin, tetracycline, and trimethoprim/sulfamethoxazole. Multilocus sequence type (MLST), serotype, average nucleotide identity (ANI), and pangenome analysis showed high genomic similarity among isolates, except for EC15 and ECV01. The EC119 plasmid contained a variety of efflux pump genes, including those encoding the acid resistance transcriptional activators (gadE, gadW, and gadX), resistance-nodulation-division-type efflux pumps (mdtE and mdtF), and a metabolite, H1 symporter (MHS) family major facilitator superfamily transporter (MNZ41_23075). Virulence genes displayed diversity, particularly EC15, whose plasmids carried genes for adherence (faeA and faeC-I), invasion (ipaH and virB), and capsule (caf1A and caf1M). This comprehensive analysis illuminates antimicrobial resistance, virulence, and plasmid dynamics in E. coli from imported shrimp and has profound implications for public health, emphasizing the need for continued surveillance and research into the evolution of these important bacterial pathogens.

Staphylococcus is a genus of Gram-positive bacteria, known to cause food poisoning and gastrointestinal illness in humans. Additionally, the emergence of methicillin-resistant S. aureus (MRSA) strains have caused major healthcare burden worldwide. Cronobacter is a group of Gram-negative bacteria, that can survive in extreme dry conditions. Cronobacter sakazakii is known to contaminate the powdered infant formula and cause life-threatening infections in neonates. Vibrio is a genus of human-pathogenic Gram-negative bacteria that can cause foodborne illness by consuming undercooked or raw seafood. Vibrio parahaemolyticus can cause serious gastrointestinal disease in humans. Thus, rapid identification of Staphylococcus spp., Cronobacter spp. and Vibrio spp. is crucial for the source tracking of contaminated food, and to measure the transmission dynamics of these bacterial pathogens causing foodborne diseases and outbreaks. This single-laboratory performance evaluation study used the VITEK MS system to evaluate the potential of MALDI-TOF MS technology for rapid identification of S. aureus-like, C. sakazakii-like, and V. parahaemolyticus-like, isolates of public health importance. A total of 226 isolates recovered from various food, environmental surveillance samples and other sources were identified by bioMérieux VITEK 2 and VITEK MS systems as Staphylococcus spp., Cronobacter spp., and Vibrio spp. Five American Type Culture Collection (ATCC) reference Gram-positive and Gram-negative bacterial isolates were also tested to complete the study. In addition, for some Staphylococcus spp. isolates whole genome sequencing (WGS), and DNA sequencing of 16S rRNA partial region was also performed for species identification. The VITEK MS system was able to provide species identification to all 96 isolates of Staphylococcus spp. and to all 29 isolates of Vibrio spp. examined with a high confidence value (99.9%). Similarly, species identification was observed for the majority of spots (245 of 303) for the 101 Cronobacter spp. isolates (∼82.0%) with a high confidence value (99.9%), and genus level identification was noticed for the rest of the Cronobacter spp. isolates (18.0%; 58 of the 303 spots) analyzed. Species identification data generated by VITEK 2 system was comparable to data obtained by VITEK MS system. The VITEK MS system is a reliable high-throughput platform that can rapidly identify Staphylococcus, Vibrio, and Cronobacter to the genus level, as well as S. aureus, C. sakazakii, V. parahaemolyticus, and other closely related foodborne isolates, and bacterial isolates from additional sources, in most cases. The VITEK MS system can be used in the rapid genus and species identification of human-pathogenic Staphylococcus spp., Cronobacter spp., and Vibrio spp. isolates.

We report the draft genome sequences of 14 fluoroquinolone-resistant Escherichia coli strains that were isolated from imported shrimp. All isolates contained multiple point mutations in the quinolone resistance-determining regions (QRDRs) and non-QRDRs of gyrA , parC , and parE genes. The data improve the understanding of fluoroquinolone resistance and indicate resistance mechanisms.

Bacteria can grow either as planktonic cells or as communities within biofilms

Staphylococcus epidermidis is a leading cause of biofilm-associated infections on implanted medical devices. During the treatment of an infection, bacterial cells inside biofilms may be exposed to sublethal concentrations of the antimicrobial agents. In the present study, the effect of subinhibitory concentrations of tigecycline (TC) on biofilms formed by S. epidermidis strain RP62A was investigated using a quantitative global proteomic technique. Sublethal concentrations of TC [1/8 (T1) and 1/4 minimum inhibitory concentration (MIC) (T2)] promoted biofilm production in strain RP62A, but 1/2 MIC TC (T3) significantly inhibited biofilm production. Overall, 413, 429, and 518 proteins were differentially expressed in biofilms grown with 1/8 (T1), 1/4 (T2), and 1/2 (T3) MIC of TC, respectively. As the TC concentration increased, the number of induced proteins in each Cluster of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway increased. The TC concentration dependence of the proteome response highlights the diverse mechanisms of adaptive responses in strain RP62A biofilms. In both COG and KEGG functional analyses, most upregulated proteins belong to the metabolism pathway, suggesting that it may play an important role in the defense of strain RP62A biofilm cells against TC stress. Sub-MIC TC treatment of strain RP62A biofilms led to significant changes of protein expression related to biofilm formation, antimicrobial resistance, virulence, quorum sensing, ABC transporters, protein export, purine/pyrimidine biosynthesis, ribosomes, and essential proteins. Interestingly, in addition to tetracycline resistance, proteins involved in resistance of various antibiotics, including aminoglycosides, antimicrobial peptides, β-lactams, erythromycin, fluoroquinolones, fusidic acid, glycopeptides, lipopeptides, mupirocin, rifampicin and trimethoprim were differentially expressed. Our study demonstrates that global protein expression profiling of biofilm cells to antibiotic pressure may improve our understanding of the mechanisms of antibiotic resistance in biofilms.