Biofilms are surface-attached groups of microbial cells encased in an extracellular matrix that are significantly less susceptible to antimicrobial agents than non-adherent, planktonic cells. Biofilm-based infections are, as a result, extremely difficult to cure. A wide range of molecular mechanisms contribute to the high degree of recalcitrance that is characteristic of biofilm communities. These mechanisms include, among others, interaction of antimicrobials with biofilm matrix components, reduced growth rates and the various actions of specific genetic determinants of antibiotic resistance and tolerance. Alone, each of these mechanisms only partially accounts for the increased antimicrobial recalcitrance observed in biofilms. Acting in concert, however, these defences help to ensure the survival of biofilm cells in the face of even the most aggressive antimicrobial treatment regimens. This review summarises both historical and recent scientific data in support of the known biofilm resistance and tolerance mechanisms. Additionally, suggestions for future work in the field are provided.

Molecular mechanisms of biofilm-based antibiotic resistance and tolerance in pathogenic bacteria

Surface-associated microbial communities, called biofilms, are present in all environments. Although biofilms play an important positive role in a variety of ecosystems, they also have many negative effects, including biofilm-related infections in medical settings. The ability of pathogenic biofilms to survive in the presence of high concentrations of antibiotics is called "recalcitrance" and is a characteristic property of the biofilm lifestyle, leading to treatment failure and infection recurrence. This review presents our current understanding of the molecular mechanisms of biofilm recalcitrance toward antibiotics and describes how recent progress has improved our capacity to design original and efficient strategies to prevent or eradicate biofilm-related infections.

https://hal-pasteur.archives-ouvertes.fr/pasteur-01370744/document

Biofilm-Related Infections: Bridging the Gap between Clinical Management and Fundamental Aspects of Recalcitrance toward Antibiotics

Increasing attention has been focused on understanding bacterial biofilms and this growth modality's relation to human disease. In this review we explore the genetic regulation and molecular components involved in biofilm formation and maturation in the context of the Gram-positive cocci, Staphylococcus aureus. In addition, we discuss diseases and host immune responses, along with current therapies associated with S. aureus biofilm infections and prevention strategies.

https://www.tandfonline.com/doi/pdf/10.4161/viru.2.5.17724

Staphylococcus aureus biofilms: properties, regulation, and roles in human disease.

Antimicrobial agents are some of the most widely, and often injudiciously, used therapeutic drugs worldwide. Important considerations when prescribing antimicrobial therapy include obtaining an accurate diagnosis of infection; understanding the difference between empiric and definitive therapy; identifying opportunities to switch to narrow-spectrum, cost-effective oral agents for the shortest duration necessary; understanding drug characteristics that are peculiar to antimicrobial agents (such as pharmacodynamics and efficacy at the site of infection); accounting for host characteristics that influence antimicrobial activity; and in turn, recognizing the adverse effects of antimicrobial agents on the host. It is also important to understand the importance of antimicrobial stewardship, to know when to consult infectious disease specialists for guidance, and to be able to identify situations when antimicrobial therapy is not needed. By following these general principles, all practicing physicians should be able to use antimicrobial agents in a responsible manner that benefits both the individual patient and the community.

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General Principles of Antimicrobial Therapy

Bacterial biofilm development as a multicellular adaptation: antibiotic resistance and new therapeutic strategies.

Objectives: This study was carried out to elucidate the role of reduced antibiotic penetration in the resistance of Staphylococcus aureus and Staphylococcus epidermidis biofilms to different antibiotics. Methods: The biofilms of S. aureus ATCC 29213 and S. epidermidis ATCC 35984 were grown on black, polycarbonate membranes (diameter, 13 mm; pore size, 0.4 mm) placed on tryptic soy agar plates at 378C for 48 h. The penetration of oxacillin, cefotaxime, amikacin, ciprofloxacin and vancomycin through the biofilms was determined by measuring the diameter of zones of growth inhibition (of S. aureus ATCC 25923, a quality control strain) on Mueller‐Hinton agar plates following diffusion of each antibiotic from an overlying antibiotic disc through the biofilm to the agar medium versus the respective control assemblies. Results: The penetration of oxacillin and cefotaxime (b-lactams) and vancomycin (a glycopeptide) was significantly reduced through S. aureus and S. epidermidis biofilms whereas that of amikacin (an aminoglycoside) and ciprofloxacin (a fluoroquinolone) was unaffected. Conclusions: The results of this study indicate that the role of reduced antibiotic penetration in the drug resistance of S. aureus and S. epidermidis biofilms may vary with the antibiotic being used.

Penetration of antibiotics through Staphylococcus aureus and Staphylococcus epidermidis biofilms

The presence of persister cells and small-colony variants (SCVs) has been associated with enhanced antibiotic resistance of many organisms in biofilms. This study investigated whether persisters and/or SCVs contribute to the antibiotic resistance of Staphylococcus aureus biofilms. A detailed dose-dependent killing of biofilms and planktonic cells with five antibiotics (oxacillin, cefotaxime, amikacin, ciprofloxacin and vancomycin) was analysed by treating them with each antibiotic at a concentration of 0-100 microg ml(-1) at 37 degrees C for 48 h. The killing of biofilm cells by all of the antibiotics showed the presence of persister cells - most cells in the population died, leaving a fraction that persisted, even at higher concentrations of the antibiotics. These persisters represented a transient resistant phenotype and reverted to a killing curve resembling that of the wild-type parent upon re-exposure to the antibiotics. SCVs were observed in biofilms only after treatment with ciprofloxacin, and these SCVs were of a transient nature. The treatment of planktonic cells with oxacillin, cefotaxime, ciprofloxacin and vancomycin killed the entire population and no persisters were detected. Transient SCVs, observed in planktonic cells following exposure to these antibiotics, were killed at higher antibiotic concentrations. The treatment of planktonic cells with amikacin yielded a small subpopulation of survivors that included persisters (at numbers significantly lower than for the biofilms) and highly resistant, stable SCVs with an increased biofilm-forming capacity in comparison with the wild-type parent. Thus the high resistance of S. aureus biofilms to multiple unrelated antibiotics is largely dependent on the presence of persister cells. Biofilms harbour a large number of persisters in comparison with planktonic cultures, which either do not harbour persisters or harbour only a small number. SCVs, although not specifically associated with S. aureus biofilms, have an increased biofilm-forming capacity and this may explain the frequent isolation of SCVs from biofilm-associated infections. The intrinsic resistance of these variants may in turn contribute to the enhanced antibiotic resistance of the biofilms thus formed.

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Role of persisters and small-colony variants in antibiotic resistance of planktonic and biofilm-associated Staphylococcus aureus: an in vitro study.

is a major global problem. Colonization rates of MRSA in the community have been reported to range from 0 to 9.2 per cent. The present study was conducted to detect S. aureus nasal colonization and prevalence of MRSA in children (5 to 15 yr) in an Indian community setting of rural, urban and semiurban slums, as also evaluation of an in-house PCR to detect MRSA. Methods: Nasal swabs from children were cultured and S. aureus isolates were processed for antibiotic susceptibility. mecA gene was studied by polymerase chain reaction (PCR) on S. aureus isolates and directly from enrichment broth aliquots inoculated with nasal swabs, at sequential time intervals. Results: The overall prevalence of S. aureus nasal colonization was 52.3 per cent and that of MRSA 3.89 per cent. CA-MRSA nasal carriage was 3.16 per cent in children without prior exposure to health care settings. PCR detection directly on nasal swabs and enrichment broth had a poor sensitivity of 60.42 per cent. Interpretation & conclusions: There was a high rate of S. aureus nasal colonization in the 5-15 yr age group and an alarming rate (3.89%) of community acquired methicillin resistant S. aureus nasal colonization in the community. PCR as a method of direct detection of MRSA from nasal samples needs further fine tuning.

A community-based study on nasal carriage of Staphylococcus aureus.

The role of Staphylococcus aureus small-colony variants (SCVs) in the pathogenesis of biofilm-associated infections remains unclear. This study investigated the mechanism behind increased biofilm-forming potential of a menadione-auxotrophic Staphylococcus aureus SCV compared with the wild-type parental strain, as recently reported by our laboratory. SCVs displayed an autoaggregative phenotype, with a greater amount of polysaccharide intercellular adhesin (PIA), significantly reduced tricarboxylic acid cycle activity and a decreased susceptibility to aminoglycosides and cell-wall inhibitors compared with wild-type. The biofilms formed by the SCV were highly structured, consisting of large microcolonies separated by channels, and contained more biomass as well as significantly more PIA than wild-type biofilms. The surface hydrophobicity of the two phenotypes was similar. Thus, the autoaggregation and increased biofilm-forming capacity of menadione-auxotrophic Staphylococcus aureus SCVs in this study was related to the enhanced production of PIA in these variants.

/pdf/enhanced-production-of-exopolysaccharide-matrix-and-biofilm-3azs324yvf.pdf

Enhanced production of exopolysaccharide matrix and biofilm by a menadione-auxotrophic Staphylococcus aureus small-colony variant.

To the Editor: Bacterial species of the genus Clostridium are anaerobic or aerotolerant, gram-positive, endospore-forming bacilli found in the soil and gut of humans and other animals. These species cause botulism, tetanus, gas gangrene, antibiotic-associated diarrhea, pseudomembranous colitis, foodborne diarrhea, and necrotic enteritis in humans and infections in other animals. Clostridium tertium, a non–exotoxin-producing, aerotolerant species, is an uncommon human pathogen. First isolated by Henry from war wounds in 1917 (1), C. tertium was recognized as a human pathogen when cases of bacteremia were reported in 1963 (2). The organism has been implicated in bacteremia (3,4), meningitis (5), septic arthritis (6), enterocolitis (7), spontaneous bacterial peritonitis (8), posttraumatic brain abscess (9), and pneumonia (4). Miller and colleagues, in a recent review of 32 cases, highlighted the role of neutropenia, intestinal mucosal injury, and exposure to β-lactam antibiotics predisposing to C. tertium bacteremia (3). C. tertium as the sole pathogen causing necrotizing fasciitis and gangrene has not been reported. We report the first two cases of necrotizing fasciitis and gangrene caused by C. tertium.

A 58-year-old man was seen at the Postgraduate Institute of Medical Education and Research, Chandigarh, India, on August 17, 2001, with two nonhealing, punched-out ulcers near the right lateral malleolus. The ulcers were approximately 2 cm x 2.5 cm in size, with necrotic margins, purulent exudate, and a foul odor. On the second day, temperature of 39.4°C and gangrene of the right leg developed. The patient also had alcoholic liver disease and non-Hodgkin lymphoma, for which he had been receiving chemotherapy for last 6 months, and he had had a 6 months’ course of antitubercular combination therapy for pulmonary tuberculosis 2 years earlier.

Peripheral blood showed a leukocyte count of 10,000/mm3 with 80% neutrophils, 11% lymphocytes, and 9% monocytes. The fasting blood glucose level was 400 mg/dL with normal electrolytes and renal function test results. Liver function tests showed a serum glutamic oxalacetic transaminase level of 80 IU/L and a serum glutamic pyruvic transaminase level of 75 IU/L. Sputum microscopy showed no acid-fast bacilli. Microscopy of necrotic tissue showed gram-positive bacilli (1–1.5 μm x 5–6 μm) with frequent oval terminal spores. Aerobic blood culture was sterile. Insulin was given to control his blood glucose level. Antimicrobial therapy included intravenous metronidazole, vancomycin, and imipenem. Skin and subcutaneous tissue debridement and fasciotomy were also undertaken.

A 40-year-old man was seen at the Postgraduate Institute on October 17, 2001, with multiple injuries of lower extremities and abdomen following a motor vehicle accident. After 24 h, the patient’s left leg and thigh turned gangrenous, and a high-grade fever (38.6°C) and an elevated leukocyte count of 14,000/mm3 (70% neutrophils, 20% lymphocytes, 10% monocytes) developed. Microscopy of necrotic tissue showed gram-positive rods (1 μm x 5 μm) with oval, terminal spores. Aerobic blood culture was sterile. Skin and subcutaneous tissue were extensively debrided. Antibiotic therapy with intravenous penicillin, metronidazole, and amikacin was instituted.

Necrotic tissues from both cases were cultured on Columbia sheep blood agar plates incubated aerobically and anaerobically (ANOXOMAT system, MART Microbiology BV, Lictenvoorde, the Netherlands) at 37°C for 24 h and 48 h, respectively. Overnight aerobic culture grew small gray colonies (<1 mm in diameter) of non–spore-forming gram-variable bacilli (1 μm x 5 μm), which, on subculture anaerobically grew gram-positive rods with oval, terminal spores. Anaerobic culture directly from specimen yielded similar spore-forming, gram-positive bacilli. The isolates were presumptively identified as Clostridium species by colony characteristics, Gram-stain morphology, and negative catalase test results; they were confirmed as C. tertium based on aerotolerance; shape and location of endospores; fermentation of glucose, lactose, maltose, and sucrose; nitrate reduction; and absence of proteolysis. Both isolates of C. tertium were susceptible in vitro to penicillin, ampicillin, vancomycin, and metronidazole.

C. tertium has been traditionally considered nonpathogenic. The organism was earlier isolated along with pathogens such as C. perfringens, C. septicum, and C. sordellii from war wounds and cases of gangrene (1). C. tertium is being increasingly reported as a human pathogen (3–9), and the strongest association has been with septicemia in patients with neutropenia and hematologic malignancies (3,4). Predisposing factors for C. tertium bacteremia include intestinal mucosal injury, neutropenia, β-lactam antibiotics (third-generation cephalosporins), cytotoxic chemotherapy, and severe liver disease, as reviewed by Miller and co-workers (3). Necrotizing fasciitis and gas gangrene caused by C. tertium as the sole pathogen have not been reported, although Miller reported necrotizing fasciitis in a patient with acute lymphocytic leukemia with C. tertium and C. septicum isolated from blood (3). The importance of isolation of C. tertium, particularly in polymicrobial cultures, is not well-established. In our report, the first patient suffered from alcoholic liver disease, had very high blood glucose levels, and was on cytotoxic chemotherapy for previous 6 months. The risk for intestinal injury is high in severe liver disease and cytotoxic chemotherapy. Intestinal mucosal compromise may potentiate translocation of C. tertium to systemic circulation and metastatic foci. The second patient had no predisposing medical history before the present episode that might have resulted in acquisition of C. tertium from the soil. Both patients had pyrexia, necrotizing fasciitis, and gangrene of a lower limb with C. tertium as the sole bacterial isolate. Neither patient had neutropenia when they were first seen. This contrasts with earlier reports of C. tertium infections (predominantly bacteremia), which usually occurred in patients with preexisting neutropenia (3). Both patients improved with penicillin or vancomycin and metronidazole, and both isolates were susceptible to these three antibiotics in vitro. Therefore, we consider both isolates to be clinically important. The pathogenesis of infection caused by C. tertium is not well understood, since the organism does not produce exotoxins. No evidence exists to correlate oxygen sensitivity with bacterial enzyme production and pathogenicity in aerotolerant clostridia. Our report adds to the list of recently emerging diseases caused by C. tertium. The growing acceptance of this organism as a human pathogen will lead to better delineation and understanding of its pathogenic potential.

http://stacks.cdc.gov/view/cdc/14326/cdc_14326_DS1.pdf

Anindita Das

Papers

Penetration of antibiotics through Staphylococcus aureus and Staphylococcus epidermidis biofilms

Role of persisters and small-colony variants in antibiotic resistance of planktonic and biofilm-associated Staphylococcus aureus: an in vitro study.

A community-based study on nasal carriage of Staphylococcus aureus.

Enhanced production of exopolysaccharide matrix and biofilm by a menadione-auxotrophic Staphylococcus aureus small-colony variant.

Clostridium tertium in necrotizing fasciitis and gangrene.