The structure and composition of bacterial communities can compromise antibiotic efficacy. For example, the secretion of β-lactamase by individual bacteria provides passive resistance for all residents within a polymicrobial environment. Here, we uncover that collective resistance can also develop via intracellular antibiotic deactivation. Real-time luminescence measurements and single-cell analysis demonstrate that the opportunistic human pathogen Streptococcus pneumoniae grows in medium supplemented with chloramphenicol (Cm) when resistant bacteria expressing Cm acetyltransferase (CAT) are present. We show that CAT processes Cm intracellularly but not extracellularly. In a mouse pneumonia model, more susceptible pneumococci survive Cm treatment when coinfected with a CAT-expressing strain. Mathematical modeling predicts that stable coexistence is only possible when antibiotic resistance comes at a fitness cost. Strikingly, CAT-expressing pneumococci in mouse lungs were outcompeted by susceptible cells even during Cm treatment. Our results highlight the importance of the microbial context during infectious disease as a potential complicating factor to antibiotic therapy.

Staphylococcus aureus is generally thought to divide in three alternating orthogonal planes over three consecutive division cycles. Although this mode of division was proposed over four decades ago, the molecular mechanism that ensures this geometry of division has remained elusive. Here we show, for three different strains, that S. aureus cells do not regularly divide in three alternating perpendicular planes as previously thought. Imaging of the divisome shows that a plane of division is always perpendicular to the previous one, avoiding bisection of the nucleoid, which segregates along an axis parallel to the closing septum. However, one out of the multiple planes perpendicular to the septum which divide the cell in two identical halves can be used in daughter cells, irrespective of its orientation in relation to the penultimate division plane. Therefore, division in three orthogonal planes is not the rule in S. aureus.

We have optimized a CRISPR interference system to facilitate gene knockdown in the gram-positive bacterial pathogen Staphylococcus aureus. For this, we used a CRISPRi system derived from Streptococcus pyogenes which requires the co-expression of the dcas9 gene encoding a catalytically inactive Cas9 protein and a customizable single guide RNA (sgRNA). In the system described in this work, dcas9 is expressed from a single copy in the chromosome of methicillin resistant S. aureus (MRSA) strains COL or JE2, under the control of a tightly regulated promoter inducible by anhydrotetracycline. The sgRNAs are expressed from a replicative plasmid under the control of a constitutively active promoter. This system enables high-efficiency, inducible, knockdown of both essential and nonessential genes and was used for the construction of the Lisbon CRISPRi mutant library (LCML) of 241 strains, in the background of JE2, containing sgRNAs targeting 209 essential genes/operons. This library allows the study of the function of essential S. aureus genes and is complementary to the Nebraska Transposon Mutant Library which consists of nearly 2000 strains, each containing a transposon insertion within a non-essential gene. Together the two libraries should facilitate the study of S. aureus pathogenesis and biology.

Aim: To test the antimicrobial effect of carbon monoxide-releasing molecules (CORMs) conjugated with azoles on different microorganisms. Methods & results: We used broth microdilution, checkerboard and cytotoxicity assays, as well as imaging, fluorescence and bioluminescence experiments to study [Re(CO)3(2,2′-bipyridyl)(Ctz)]+ (also known as ReBpyCtz). ReBpyCtz exhibits a low minimum inhibitory concentration value, increases the intracellular formation of reactive oxygen species and causes significant alterations on Staphylococcus aureus‘s membrane. ReBpyCtz is active against fungi, having a more prolonged fungicidal effect on Candida glabrata than clotrimazole and is selectively active on blood-stage malaria parasites, at a concentration that is not toxic to kidney epithelial cells. Conclusion: Conjugated CORMs have the potential to be active against different types of pathogens, thus constituting a promising class of broad-spectrum antimicrobials.