Complex interbacterial interactions in mixed biofilms as a key determinant of their antimicrobial treatment efficacy

Biofilms are formed by closely adjacent microorganisms embedded into an extracellular matrix this way providing them with strong protection from antimicrobials, which is often further reinforced in polymicrobial biofilms. Despite of the well-known antagonistic interactions between S. aureus and P. aeruginosa, the most common pathogens causing various nosocomial infections, they often form mixed consortia characterized by increased pathogenicity and delayed recovery in comparison with single species infections. Here we show that, while S. aureus could successfully avoid a number of antimicrobials by embedding into the biofilm matrix of P. aeruginosa despite of their antagonism, the very same consortium was characterized by 10–fold higher susceptibility to broad-spectrum antimicrobials compared to monocultures. Moreover, quantitatively similar increase in antimicrobials susceptibility could be achieved when P. aeruginosa was introduced into S. aureus biofilm, compared to S. aureus monoculture. In a reverse experiment, intervention of S. aureus into the mature P. aeruginosa biofilm significantly increased the efficacy of ciprofloxacin against P. aeruginosa. A broader perspective is provided by antagonistic bacteria intervention into already preformed monoculture biofilms leading to the considerable enhancement of their antibiotic susceptibility. We believe that this approach has a strong potential of further development towards innovative treatment of biofilm-associated infections such as transplantation of the skin residential microflora to the wounds and ulcers infected with nosocomial pathogens to speed up their microbial decontamination. Author summary Biofilms formation is one of the key mechanisms providing pathogenic bacteria with extreme resistance to antimicrobials. On the S. aureus and P. aeruginosa mixed culture model we show explicitly that antimicrobials efficacy against bacteria in mixed biofilms differs considerably from monoculture biofilms. From the one hand, S. aureus avoids vancomycin and ampicillin by the rearrangements to the lower layers of the P. aeruginosa biofilm matrix. On the other hand, in the same consortium susceptibility to ciprofloxacin and aminoglycosides increases nearly 10– fold compared to monocultures. This finding allowed suggesting that intervention of antagonistic bacteria into already preformed monoculture biofilms could be used as an innovative approach to their treatment by increasing their antibiotic susceptibility. Thus, by introducing P. aeruginosa into preformed S. aureus biofilm, susceptibility of S. aureus to aminoglycosides was increased 4-fold, compared to monoculture. The intervention of S. aureus into the mature P. aeruginosa biofilm significantly increased the efficacy of ciprofloxacin against P. aeruginosa. We believe that this approach has a strong potential of further development towards innovative treatment of biofilm-associated infections such as introduction of the skin residential microflora to the wounds and ulcers infected with nosocomial pathogens to speed up their microbial decontamination.


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While it is known that S. aureus and P. aeruginosa exhibit rather antagonistic relationship 88 [22,23], several studies reported their mutual association in acute and chronic wounds embedded 89 in a mixed biofilm [8, 24-28], with S. aureus typically residing on the wound surface, whereas 90 6 P. aeruginosa being rather observed in the deep layers [15,[28][29][30][31] able to penetrate into the preformed biofilm of the other bacterium (Fig 1). Irrespective of which 146 bacterium initially preformed the biofilm and which one was added later, the ratio of their CFUs 147 in the biofilm after 24 h cultivation remained around 1:10 with the prevalence of the first biofilm 148 former (Fig 1 A and B), and was 1:1 when both bacteria were inoculated simultaneously (Fig 1   149 C). Therefore in the following experiments simultaneous inoculation of both bacteria was used to 150 obtain their mixed biofilm.

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Next to analyze the biofilm structure and cells distribution in the matrix, the S. aureus -152 P. aeruginosa mixed biofilm was grown in imaging cover slips, stained with ViaGram TM Red + to 153 differentiate between S. aureus and P. aeruginosa followed by their analysis with confocal laser 154 scanning microscopy. To estimate the viability of the cells, SYTO9/propidium iodide staining 155 was also performed, as the ViaGram TM Red + staining requires buffer change that disturbs the 156 biofilm structure. Both S. aureus and P. aeruginosa formed 20-25 m-thick biofilms when 157 growing as monocultures (Fig 2A, B). While the mixed biofilm was of similar thickness, it 158 appeared more rigid in comparison with monoculture ones (Fig 2C). Interestingly, in the mixed 159 biofilm, S. aureus was distributed unevenly and appeared as cell clumps, apparently as so-called 160 small colony variants (SCV) embedded in the biofilm matrix (see white arrow in Fig 2C). By 161 9 using differential staining of S. aureus and P. aeruginosa (Fig 3) we have also analyzed the 162 distributions of S. aureus (red-stained) and P. aeruginosa (blue-stained) over the biofilm layers 163 and evaluated their relative fractions in each layer. In agreement with earlier data, S. aureus 164 tended to distribute in the upper layers of the biofilm, while P. aeruginosa dominated in its lower 165 layers (see Fig 3A, C). The fraction of non-viable cells in the mixed biofilm was just slightly 166 higher than in corresponding monoculture biofilms (compare Fig 2 A Table 1 for MBC values).

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The distribution of bacteria in the mixed biofilm layers was also assessed by differential 294 staining of S. aureus and P. aeruginosa by the ViaGram TM Red + (Fig 6). In marked contrast to    (Fig S4 and S6). These data suggest that under anti-biofilm 335 or antimicrobial treatment conditions S. aureus changes its preferred topical localizations by 336 hiding in the lower layers of mixed biofilm formed by another bacterium like P. aeruginosa 337 insensitive to most antimicrobials thereby increasing its resistance to the treatment.

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Next, we investigated the effect of broad-spectrum antimicrobials such as ciprofloxacin, 339 amikacin and gentamycin which are active against both S. aureus and P. aeruginosa (see Table   340 1). In contrast to the previous group of antimicrobials, in monoculture high concentrations of 341 ciprofloxacin efficiently eradicated even the biofilm-embedded P. aeruginosa (Fig 7 B).

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Interestingly, when the mixed biofilm was treated, nearly 10-fold lower concentration of 343 antimicrobial was required to obtain similar reduction of P. aeruginosa CFUs in the biofilm.

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The CLSM analysis of S. aureus and P. aeruginosa monoculture and mixed biofilms 378 treated with Ciprofloxacin confirmed the CFUs counting data. In particular, while 8×MBC did 379 not affect either S. aureus or P. aeruginosa cells in monoculture biofilms (Fig 7E, F), in the 380 mixed biofilm P. aeruginosa was identified as non-viable, although S. aureus remained partially 381 alive (Fig 7 G). In marked contrast, repression of the S. aureus biofilm production by F105 led to 382 a reversal with most P. aeruginosa cells green-stained while S. aureus identified as non-viable in 383 mixed culture (Fig 7 J).

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The distribution of bacteria in the mixed biofilm layers under treatment with 385 ciprofloxacin was also assessed by differential staining of S. aureus and P. aeruginosa using 386 ViaGram TM Red + (Fig 9). In contrast to vancomycin treatment, here S. aureus dominated in the 387 upper layers of the mixed biofilm (compare Fig 6 and 9 A and C) and remain alive, while 388 P. aeruginosa were presumably dead (See Fig S5, S7, S8) suggesting no reversal protection of     Table 1). Additionally, we also simulated the biofilm-preventing treatment with earlier

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To determine the MBC of antimicrobials the CFU/mL were further evaluated in the 590 culture liquid from those wells without visible growth. 10 μl of the culture liquid from the wells 591 with no visible growth were inoculated into 3ml of LB broth followed by cultivation for 24h.