Adhesion of Klebsiella oxytoca to bladder or lung epithelial cells is promoted by the presence of other opportunistic pathogens.

The intestinal and respiratory tracts of healthy individuals serve as habitats for a diverse array of microorganisms, among which Klebsiella oxytoca holds significance as a causative agent in numerous community- and hospital-acquired infections, often manifesting in polymicrobial contexts. In specific circumstances, K. oxytoca, alongside other constituents of the gut microbiota, undergoes translocation to distinct physiological niches. In these new environments, it engages in close interactions with other microbial community members. As this interaction may progress to co-infection where the virulence of involved pathogens may be promoted and enhance disease severity, we investigated how K. oxytoca affects the adhesion of commonly co-isolated bacteria and vice versa during co-incubation of different biotic and abiotic surfaces. Co-incubation was beneficial for the adhesion of at least one of the two co-cultured strains. K. oxytoca enhanced the adhesion of other enterobacteria strains to polystyrene and adhered more efficiently to bladder or lung epithelial cell lines in the presence of most enterobacteria strains and S. aureus. This effect was accompanied by bacterial coaggregation mediated by carbohydrate-protein interactions occurring between bacteria. These interactions occur only in sessile, but not planktonic populations, and depend on the features of the surface. The data are of particular importance for the risk assessment of the urinary and respiratory tract infections caused by K. oxytoca, including those device-associated. In this paper, we present the first report on K. oxytoca ability to acquire increased adhesive capacities on epithelial cells through interactions with common causal agents of urinary and respiratory tract infections.

Phage-antibiotic combinations to control Pseudomonas aeruginosa-Candida two-species biofilms.

Phage-antibiotic combinations to treat bacterial infections are gaining increased attention due to the synergistic effects often observed when applying both components together. Most studies however focus on a single pathogen, although in many clinical cases multiple species are present at the site of infection. The aim of this study was to investigate the anti-biofilm activity of phage-antibiotic/antifungal combinations on single- and dual-species biofilms formed by P. aeruginosa and the fungal pathogen Candida albicans. The Pseudomonas phage Motto in combination with ciprofloxacin had significant anti-biofilm activity. We then compared biofilms formed by P. aeruginosa alone with the dual-species biofilms formed by bacteria and C. albicans. Here, we found that the phage together with the antifungal fluconazole was active against 6-h-old dual-species biofilms but showed only negligible activity against 24-h-old biofilms. This study lays the first foundation for potential therapeutic approaches to treat co-infections caused by bacteria and fungi using phage-antibiotic combinations.

Xanthone Derivatives Enhance the Therapeutic Potential of Neomycin against Polymicrobial Gram-Negative Bacterial Infections.

Gram-negative bacterial infections are difficult to manage as many antibiotics are ineffective owing to the presence of impermeable bacterial membranes. Polymicrobial infections pose a serious threat due to the inadequate efficacy of available antibiotics, thereby necessitating the administration of antibiotics at higher doses. Antibiotic adjuvants have emerged as a boon as they can augment the therapeutic potential of available antibiotics. However, the toxicity profile of antibiotic adjuvants is a major hurdle in clinical translation. Here, we report the design, synthesis, and biological activities of xanthone-derived molecules as potential antibiotic adjuvants. Our SAR studies witnessed that the p-dimethylamino pyridine-derivative of xanthone (X8) enhances the efficacy of neomycin (NEO) against Escherichia coli and Pseudomonas aeruginosa and causes a synergistic antimicrobial effect without any toxicity against mammalian cells. Biochemical studies suggest that the combination of X8 and NEO, apart from inhibiting protein synthesis, enhances the membrane permeability by binding to lipopolysaccharide. Notably, the combination of X8 and NEO can disrupt the monomicrobial and polymicrobial biofilms and show promising therapeutic potential against a murine wound infection model. Collectively, our results unveil the combination of X8 and NEO as a suitable adjuvant therapy for the inhibition of the Gram-negative bacterial infections.

Enterococci facilitate polymicrobial infections.

Enterococci are ubiquitous members of the gut microbiota in human beings and animals and are among the most important nosocomial organisms. Due to their opportunistic pathogenicity, enterococci are referred to as pathobionts and play decisive roles in a diverse array of polymicrobial infections. Enterococci can promote the colonization, pathogenesis, and persistence of various pathogens, compromise the efficacy of drugs, and pose a severe threat to public health. Most current treatments tend to focus on the sole pathogenic bacteria, with insufficient attention to the driving role of enterococci. In this review, we summarize the characteristics of enterococci in infections, the factors facilitating their outgrowth, as well as the sites and types of enterococci-associated polymicrobial infections. We present an overview of the underlying mechanisms of enterococci-mediated pathogenesis in polymicrobial infections. Furthermore, we discuss alternative strategies and potential intervention approaches to restrict such infections, shedding light on the discovery and development of new therapies against polymicrobial infections.

Antimicrobial Susceptibility Profiles of Acinetobacter baumannii Strains, Isolated from Clinical Cases of Companion Animals in Greece.

Acinetobacter baumannii-calcoaceticus (Abc) Complex bacteria are troublesome nosocomial pathogens in human medicine, especially during the last 30 years. Recent research in veterinary medicine also supports its emergence as an animal pathogen. However, relevant data are limited. In this study, we obtained 41 A. baumannii isolates from clinical samples of canine and feline origin collected in veterinary clinics in Greece between 2020 and 2023. Biochemical identification, antimicrobial susceptibility testing, molecular identification and statistical analysis were performed. Most of the samples were of soft tissue and urine origin, while polymicrobial infections were recorded in 29 cases. Minocycline was the most effective in vitro antibiotic, whereas high resistance rates were detected for almost all the agents tested. Notably, 20 isolates were carbapenem resistant and 19 extensively drug resistant (XDR). This is the first report of canine and feline infections caused by Abc in Greece. The results create concerns regarding the capability of the respective bacteria to cause difficult-to-treat infections in pets and persist in veterinary facilities through hospitalized animals, contaminated equipment, and surfaces. Moreover, the prevalence of highly resistant strains in companion animals constitutes a public health issue since they could act as a reservoir, contributing to the spread of epidemic clones in a community.

Rapid and strain-specific resistance evolution of Staphylococcus aureus against inhibitory molecules secreted by Pseudomonas aeruginosa.

IMPORTANCE: Polymicrobial infections are common. In chronic infections, the different pathogens may repeatedly interact, which could spur evolutionary dynamics with pathogens adapting to one another. Here, we explore the potential of Staphylococcus aureus to adapt to its competitor Pseudomonas aeruginosa. These two pathogens frequently co-occur, and P. aeruginosa is seen as the dominant species being able to displace S. aureus. We studied three different S. aureus strains and found that all became quickly resistant to inhibitory compounds secreted by P. aeruginosa. Our experimental evolution revealed strains-specific adaptations with three main factors contributing to resistance evolution: (i) overproduction of staphyloxanthin, a molecule protecting from oxidative stress; (ii) the formation of small colony variants also protecting from oxidative stress; and (iii) alterations of membrane transporters possibly reducing toxin uptake. Our results show that species interactions can change over time potentially favoring species co-existence, which in turn could affect disease progression and treatment options.

Draft genome sequences of 12 Escherichia coli co-isolated with Enterococcus spp. from dogs with polybacterial bacteriuria at a veterinary hospital.

Escherichia coli are frequently co-isolated with Enterococcus spp. from urine cultures of dogs with urinary tract infections (UTIs). Uropathogenic E. coli (UPEC) are augmented by Enterococcus in polymicrobial UTIs. We report the draft genome sequences of 12 UPEC co-isolated with Enterococcus spp. from canine urinary tract infections.

Multipathogen infections and multifactorial pathogenesis involved in noble pen shell (Pinna nobilis) mass mortality events: Background and current pathologic approaches.

Disease outbreaks in several ecologically or commercially important invertebrate marine species have been reported in recent years all over the world. Mass mortality events (MMEs) have affected the noble pen shell (Pinna nobilis), causing its near extinction. Our knowledge of the dynamics of diseases affecting this species is still unclear. Early studies investigating the causative etiological agent focused on a novel protozoan parasite, Haplosporidium pinnae, although further investigations suggested that concurrent polymicrobial infections could have been pivotal in some MMEs, even in the absence of H. pinnae. Indeed, moribund specimens collected during MMEs in Italy, Greece, and Spain demonstrated the presence of a bacteria from within the Mycobacterium simiae complex and, in some cases, species similar to Vibrio mediterranei. The diagnostic processes used for investigation of MMEs are still not standardized and require the expertise of veterinary and para-veterinary pathologists, who could simultaneously evaluate a variety of factors, from clinical signs to environmental conditions. Here, we review the available literature on mortality events in P. nobilis and discuss approaches to define MMEs in P. nobilis. The proposed consensus approach should form the basis for establishing a foundation for future studies aimed at preserving populations in the wild.

Genetic Factors That Contribute to Antibiotic Resistance through Intrinsic and Acquired Bacterial Genes in Urinary Tract Infections.

The overprescribing and misuse of antibiotics have led to the rapid development of multidrug-resistant bacteria, such as those that cause UTIs. UTIs are the most common outpatient infections and are mainly caused by Escherichia coli and Klebsiella spp., although some Gram-positive bacteria, such as Pseudomonas aeruginosa, have been isolated in many cases. The rise of antimicrobial-resistant bacteria is a major public health concern, as it is predicted to lead to increased healthcare costs and poor patient outcomes and is expected to be the leading cause of global mortality by 2050. Antibiotic resistance among bacterial species can arise from a myriad of factors, including intrinsic and acquired resistance mechanisms, as well as mobile genetic elements, such as transposons, integrons, and plasmids. Plasmid-mediated resistance is of major concern as drug-resistance genes can quickly and efficiently spread across bacterial species via horizontal gene transfer. The emergence of extended-spectrum ß-lactamases (ESBLs) such as NDM-1, OXA, KPC, and CTX-M family members has conferred resistance to many commonly used antibiotics in the treatment of UTIs, including penicillins, carbapenems, cephalosporins, and sulfamethoxazole. This review will focus on plasmid-mediated bacterial genes, especially those that encode ESBLs, and how they contribute to antibiotic resistance. Early clinical detection of these genes in patient samples will provide better treatment options and reduce the threat of antibiotic resistance.

Parvimonas micra a new potential pathogen in hospitalized patients: a case series from 2015-2022.

Parvimonas micra isolations are usually part of polymicrobial infections and the pathogenic role of this microrganism is still debated. We describe here a large series of hospitalized patients diagnosed with Parvimonas micra infections and discuss the clinical and therapeutic management and the outcome of these infections.

Efflux, Signaling and Warfare in a Polymicrobial World.

The discovery void of antimicrobial development has occurred at a time when the world has seen a rapid emergence and spread of antimicrobial resistance, the 'perfect storm' as it has often been described. While the discovery and development of new antibiotics has continued in the research sphere, the pipeline to clinic has largely been fed by derivatives of existing classes of antibiotics, each prone to pre-existing resistance mechanisms. A novel approach to infection management has come from the ecological perspective whereby microbial networks and evolved communities already possess small molecular capabilities for pathogen control. The spatiotemporal nature of microbial interactions is such that mutualism and parasitism are often two ends of the same stick. Small molecule efflux inhibitors can directly target antibiotic efflux, a primary resistance mechanism adopted by many species of bacteria and fungi. However, a much broader anti-infective capability resides within the action of these inhibitors, borne from the role of efflux in key physiological and virulence processes, including biofilm formation, toxin efflux, and stress management. Understanding how these behaviors manifest within complex polymicrobial communities is key to unlocking the full potential of the advanced repertoires of efflux inhibitors.

Infections in Glucose-6-Phosphate Dehydrogenase G6PD-Deficient Patients; Predictors for Infection-Related Mortalities and Treatment Outcomes.

Disturbances in the count or maturity of blood cells weaken their microbial defensive capacity and render them more susceptible to infections. Glucose-6-phosphate deficient patients are affected by a genetic disease that affects cell integrity with increased liability to infections and death. We aimed to investigate the risk factors for infection mortality in this patient population. We retrospectively examined the records of G6PD adult patients with confirmed infections and collected data related to demographics, infections (pathogens, types, and treatment regimens) in addition to mortality and length of stay outcomes. Data were statistically analyzed using R Programming language to identify contributing factors to mortality and treatment regimens association with outcomes. Records of 202 unique patients over 5 years were included, corresponding to 379 microbiologically and clinically confirmed infections. Patients > 60 years [p = 0.001, OR: 5.6], number of comorbidities 4 (2-5) [p < 0.001, OR: 1.8], patients needed blood transfusion [p = 0.003, OR: 4.3]. Respiratory tract infections [p = 0.037, OR: 2.28], HAIs [p = 0.002, OR: 3.9], polymicrobial infections [p = 0.001, OR: 10.9], and concurrent infection Gram-negative [p < 0.001, OR: 7.1] were significant contributors to 28-day mortality. The history of exposure to many antimicrobial classes contributed significantly to deaths, including ß-lactam/ß-lactamase [p = 0.002, OR: 2.5], macrolides [p = 0.001, OR: 3.34], and ß-lactams [p = 0.012, OR: 2.0]. G6PD patients are a unique population that is more vulnerable to infections. Prompt and appropriate antimicrobial therapy is warranted to combat infections. A strict application of stewardship principles (disinfection, shortening the length of stay, and controlling comorbid conditions) may be beneficial for this population. Finally, awareness of the special needs of this patient group may improve treatment outcomes.

Myxinidin-Derived Peptide against Biofilms Caused by Cystic Fibrosis Emerging Pathogens.

Chronic lung infections in cystic fibrosis (CF) patients are triggered by multidrug-resistant bacteria such as Pseudomonas aeruginosa, Achromobacter xylosoxidans, and Stenotrophomonas maltophilia. The CF airways are considered ideal sites for the colonization and growth of bacteria and fungi that favor the formation of mixed biofilms that are difficult to treat. The inefficacy of traditional antibiotics reinforces the need to find novel molecules able to fight these chronic infections. Antimicrobial peptides (AMPs) represent a promising alternative for their antimicrobial, anti-inflammatory, and immunomodulatory activities. We developed a more serum-stable version of the peptide WMR (WMR-4) and investigated its ability to inhibit and eradicate C. albicans, S. maltophilia, and A. xylosoxidans biofilms in both in vitro and in vivo studies. Our results suggest that the peptide is able better to inhibit than to eradicate both mono and dual-species biofilms, which is further confirmed by the downregulation of some genes involved in biofilm formation or in quorum-sensing signaling. Biophysical data help to elucidate its mode of action, showing a strong interaction of WMR-4 with lipopolysaccharide (LPS) and its insertion in liposomes mimicking Gram-negative and Candida membranes. Our results support the promising therapeutic application of AMPs in the treatment of mono- and dual-species biofilms during chronic infections in CF patients.

Evaluation of InTray Cassettes Directly from Blood Cultures for the Diagnosis of Sepsis in Clinical Bacteriology Laboratories as an Alternative to Classic Culture Media.

Culture media is fundamental in clinical bacteriology for the detection and isolation of bacterial pathogens. However, in-house media preparation could be challenging in low-resource settings. InTray® cassettes (Biomed Diagnostics) could be a valid alternative as they are compact, ready-to-use media preparations. In this study, we evaluate the use of two InTray media as a subculture alternative for the diagnosis of bloodstream infections: the InTray® Müller-Hinton (MH) chocolate and the InTray® Colorex™ Screen. The InTray MH chocolate was evaluated in 2 steps: firstly, using simulated positive blood cultures (reference evaluation study), and secondly, using positive blood cultures from a routine clinical laboratory (clinical evaluation study). The Colorex Screen was tested using simulated poly-microbial blood cultures. The sensitivity and specificity of the InTray MH chocolate were respectively 99.2% and 90% in the reference evaluation study and 97.1% and 88.2% in the clinical evaluation study. The time to detection (TTD) was ≤20 h in most positive blood cultures (99.8% and 97% in the two studies, respectively). The InTray® MH Chocolate agar showed good performance when used directly from clinical blood cultures for single bacterial infections. However, mixed flora is more challenging to interpret on this media than on Colorex™ Screen, even for an experienced microbiologist.

Advances in bacteriophage-mediated strategies for combating polymicrobial biofilms.

Bacteria and fungi tend to coexist within biofilms instead of in planktonic states. Usually, such communities include cross-kingdom microorganisms, which make them harder to remove from abiotic surfaces or infection sites. Additionally, the produced biofilm matrix protects embedded microorganisms from antibiotics, disinfectants, or the host immune system. Therefore, classic therapies based on antibiotics might be ineffective, especially when multidrug-resistant bacteria are causative factors. The complexities surrounding the eradication of biofilms from diverse surfaces and the human body have spurred the exploration of alternative therapeutic modalities. Among these options, bacteriophages and their enzymatic counterparts have emerged as promising candidates, either employed independently or in synergy with antibiotics and other agents. Phages are natural bacteria killers because of mechanisms of action that differ from antibiotics, phages might answer worldwide problems with bacterial infections. In this review, we report the attempts to use bacteriophages in combating polymicrobial biofilms in in vitro studies, using different models, including the therapeutical use of phages. In addition, we sum up the advantages, disadvantages, and perspectives of phage therapy.

NGS in the clinical microbiology settings.

We hypothesized that targeted NGS sequencing might have an advantage over Sanger sequencing, especially in polymicrobial infections. The study included 55 specimens from 51 patients. We compared targeted NGS to Sanger sequencing in clinical samples submitted for Sanger sequencing. The overall concordance rate was 58% (32/55) for NGS vs. Sanger. NGS identified 9 polymicrobial and 2 monomicrobial infections among 19 Sanger-negative samples and 8 polymicrobial infections in 11 samples where a 16S gene was identified by gel electrophoresis, but could not be mapped to an identified pathogen by Sanger. We estimated that NGS could have contributed to patient management in 6/18 evaluated patients and thus has an advantage over Sanger sequencing in certain polymicrobial infections.

Tachyplesin I Analogue Peptide as an Effective Antimicrobial Agent against Candida albicans-Staphylococcus aureus Poly-Biofilm Formation and Mixed Infection.

Microbial biofilms are difficult to tackle in many infectious diseases. Candida albicans and Staphylococcus aureus are prevalent symbiotic strains in polymicrobial biofilms, which showed enhanced antimicrobial resistance and made identifying effective treatment techniques more difficult. The antibiofilm abilities of tachplesin I analogue peptide (TP11A) and tachplesin I were investigated quantitatively in this study. Both inhibited C. albicans monomicrobial, S. aureus monomicrobial, and C. albicans-S. aureus polymicrobial biofilms quite well. TP11A suppressed the biofilm- and virulence-related genes of C. albicans (hwp 1) and S. aureus (ica A, fnb B, agr A, hla, nor A, and sig B) in the mixed biofilm, according to quantitative reverse transcription polymerase chain reaction analysis. We created an injectable thermosensitive in situ PLEL@TP11A gel system by simply adding TP11A into poly(d,l-lactide)-poly(ethylene glycol)-poly(d,l-lactide) (PLEL). Using C. albicans-S. aureus mixed infected wound models of mice, the in vivo therapeutic effect of TP11A and PLEL@TP11A in polymicrobial infections was investigated. The findings revealed that TP11A and PLEL@TP11A could efficiently reduce bacterial and fungal burden in wound infections, as well as accelerated wound healing. Based on above findings, TP11A might be an effective antimicrobial against C. albicans-S. aureus poly-biofilm formation and mixed infection.

Engineering Antimicrobial Polymer Nanocomposites: In Situ Synthesis, Disruption of Polymicrobial Biofilms, and In Vivo Activity.

The increasing incidence of microbial infections and a limited arsenal of effective antibacterial and antifungal agents have entailed the need for new broad-spectrum therapeutics. Polymer-inorganic nanocomposites have emerged as an integral choice of antimicrobials but are limited by complicated synthesis, narrow-spectrum activity, and poor in vivo efficacy. Herein, chloride counterions of a nontoxic, moderately antibacterial polymer have been explored for in situ nanoprecipitation-based synthesis of water-soluble polymer-silver chloride nanocomposites. With the controlled release of silver ions, the nanocomposites were highly active against multidrug-resistant bacteria as well as fluconazole-resistant fungi. Alongside the elimination of metabolically inactive bacterial cells, the nanocomposites disrupted polymicrobial biofilms, unlike antibiotics and only silver-based ointments. This underlined the role of the engineered composite design, where the polymer interacted with the biofilm matrix, facilitating the penetration of nanoparticles to kill microbes. Further, the nanocomposite diminished Pseudomonas aeruginosa burden in mice skin infection (>99.9%) with no dermal toxicity proving its potential for clinical translation.

Antibacterial Designs for Implantable Medical Devices: Evolutions and Challenges.

The uses of implantable medical devices are safer and more common since sterilization methods and techniques were established a century ago; however, device-associated infections (DAIs) are still frequent and becoming a leading complication as the number of medical device implantations keeps increasing. This urges the world to develop instructive prevention and treatment strategies for DAIs, boosting the studies on the design of antibacterial surfaces. Every year, studies associated with DAIs yield thousands of publications, which here are categorized into four groups, i.e., antibacterial surfaces with long-term efficacy, cell-selective capability, tailored responsiveness, and immune-instructive actions. These innovations are promising in advancing the solution to DAIs; whereas most of these are normally quite preliminary "proof of concept" studies lacking exact clinical scopes. To help identify the flaws of our current antibacterial designs, clinical features of DAIs are highlighted. These include unpredictable onset, site-specific incidence, and possibly involving multiple and resistant pathogenic strains. The key point we delivered is antibacterial designs should meet the specific requirements of the primary functions defined by the "intended use" of an implantable medical device. This review intends to help comprehend the complex relationship between the device, pathogens, and the host, and figure out future directions for improving the quality of antibacterial designs and promoting clinical translations.