The increasing prevalence of antimicrobial resistance and the limited availability of new antimicrobial agents have created an urgent need for new approaches to combat these issues. One such approach involves reevaluating the use of old antibiotics to ensure their appropriate usage and maximize their effectiveness, as older antibiotics could help alleviate the burden on newer agents. An example of such an antibiotic is chloramphenicol (CHL), which is rarely used due to its hematological toxicity. In the current study, we employed a previously published transposon mutant library in MG1655/pTF2::bla_CTX-M-1, containing over 315,000 unique transposon insertions, to identify the genetic factors that play an important role during growth in the presence of CHL. The list of conditionally essential genes, collectively referred to as the secondary resistome (SR), included 67 genes. To validate our findings, we conducted gene knockout experiments on six genes: arcA, hfq, acrZ, cls, mdfA, and nlpI. Deleting these genes resulted in increased susceptibility to CHL as demonstrated by MIC estimations and growth experiments, suggesting that targeting the products encoded from these genes may reduce the dose of CHL needed for treatment and hence reduce the toxicity associated with CHL treatment. Thus, the gene products are indicated as targets for antibiotic adjuvants to favor the use of CHL in modern medicine.

Due to the rapid spread of CTX-M type ESBLs, the rate of resistance to third-generation cephalosporin has increased among Gram-negative bacteria, especially in Escherichia coli, and there is a need to find ways to re-sensitize ESBL E. coli to cephalosporin treatment. A previous study showed that genes involved in protein synthesis were significantly up-regulated in the presence of subinhibitory concentration of cefotaxime (CTX) in a CTX-M-1-producing E. coli. In this study, the interaction between CTX and gentamicin (GEN), targeting protein synthesis, was evaluated in MG1655/pTF2, and the MIC of CTX was strongly reduced (128-fold) in the presence of this combnation therapy. Since the underlying mechanism behind this synergy is not known, we constructed a saturated transposon mutant library in MG1655/pTF2::bla_CTX-M-1 containing 315,925 unique transposon insertions to measure mutant depletion upon exposure to CTX, GEN, and combination treatment of CTX and GEN by Transposon Directed Insertion-site Sequencing (TraDIS). We identified 57 genes that were depleted (log₂FC ≤ −2 and with q.value ≤ 0.01) during exposure to CTX, 18 for GEN, and 31 for combination treatment of CTX and GEN. For validation, we deleted eight genes that were either uniquely identified in combination treatment, overlapped with monotherapy of GEN, or were shared between combination treatment and monotherapy with CTX and GEN. Of these genes, we found that the inactivation of dnaK, mnmA, rsgA, and ybeD increased the efficacy of both CTX and GEN treatment, the inactivation of cpxR and yafN increased the efficacy of only CTX, and the inactivation of mnmA, rsgA, and ybeD resulted in increased synergy between CTX and GEN. Thus, the study points to putative targets for helper drugs that can restore susceptibility to these important drugs, and it indicates that genes involved in protein synthesis are essential for the synergy between these two drugs. In summary, the study identified mutants that sensitize ESBL-producing E. coli to CTX and a combination of CTX and GEN, and it increased our understanding of the mechanism behind synergy between β-lactam and aminoglycoside drugs. This forms a framework for developing new strategies to combat infections caused by resistant bacteria.

Background: Therapeutic strategies for methicillin-resistant Staphylococcus aureus (MRSA) are increasingly limited due to the ability of the pathogen to evade conventional treatments such as vancomycin and daptomycin. This challenge has shifted the focus towards novel strategies, including the resensitization of β-lactams, which are still used as first-line treatments for methicillin-susceptible Staphylococcus aureus (MSSA). To achieve this, it is essential to identify the secondary resistome associated with the clinically relevant β-lactam antibiotics. Methods: Transposon-Directed Insertion Site Sequencing (TraDIS) was employed to assess conditional essentiality by analyzing the depletion of mutants from a highly saturated transposon library of MRSA USA300 JE2 exposed to ½ minimal inhibitory concentration (MIC) of oxacillin or cefazolin. Results: TraDIS analysis led to the identification of 52 shared fitness genes involved in β-lactam resistance that are primarily linked to cell wall metabolism and regulatory systems. Among these, both known resistance factors and novel conditionally essential genes were highlighted. As proof of concept, transposon mutants corresponding to nine genes (sagB, SAUSA300_0657, SAUSA300_0957, SAUSA300_1683, SAUSA300_1964, SAUSA300_1966, SAUSA300_1967, SAUSA300_1692, and mazF) were grown in the presence of β-lactam antibiotics and their MICs were determined. All mutants showed significantly reduced resistance to β-lactam antibiotics. Conclusions: This comprehensive genome-wide investigation provides novel insights into the resistance mechanisms of β-lactam antibiotics, and suggests potential therapeutic targets for combination therapies with helper drugs.

Among genes present in all group A streptococci (GAS), those encoding M-fibril and T-pilus proteins display the highest levels of sequence diversity, giving rise to the two primary serological typing schemes historically used to define strain. A new genotyping scheme for the pilin adhesin and backbone genes is developed and, when combined with emm typing, provides an account of the global GAS strain population. Cluster analysis based on nucleotide sequence similarity assigns most T-serotypes to discrete pilin backbone sequence clusters, yet the established T-types correspond to only half the clusters. The major pilin adhesin and backbone sequence clusters yield 98 unique combinations, defined as “pilin types.” Numerous horizontal transfer events that involve pilin or emm genes generate extensive antigenic and functional diversity on the bacterial cell surface and lead to the emergence of new strains. Inferred pilin genotypes applied to a meta-analysis of global population-based collections of pharyngitis and impetigo isolates reveal highly significant associations between pilin genotypes and GAS infection at distinct ecological niches, consistent with a role for pilin gene products in adaptive evolution. Integration of emm and pilin typing into open-access online tools ( pubmlst.org ) ensures broad utility for end-users wanting to determine the architecture of M-fibril and T-pilus genes from genome assemblies. IMPORTANCE: Precision in defining the variant forms of infectious agents is critical to understanding their population biology and the epidemiology of associated diseases. Group A Streptococcus (GAS) is a global pathogen that causes a wide range of diseases and displays a highly diverse cell surface due to the antigenic heterogeneity of M-fibril and T-pilus proteins which also act as virulence factors of varied functions. emm genotyping is well-established and highly utilized, but there is no counterpart for pilin genes. A global GAS collection provides the basis for a comprehensive pilin typing scheme, and online tools for determining emm and pilin genotypes are developed. Application of these tools reveals the expansion of structural-functional diversity among GAS via horizontal gene transfer, as evidenced by unique combinations of surface protein genes. Pilin and emm genotype correlations with superficial throat vs skin infection provide new insights on the molecular determinants underlying key ecological and epidemiological trends.