Staphylococcal cassette chromosome mec (SCC mec ) typing, in combination with genotyping of the Staphylococcus aureus chromosome, has become essential for defining methicillin-resistant S. aureus (MRSA) clones in epidemiological studies. We have developed a convenient system for SCC mec type assignment. The system consists of six multiplex PCRs (M-PCRs) for identifying the ccr gene complex ( ccr ), the mec gene complex ( mec ), and specific structures in the junkyard (J) regions: M-PCR with primer set 1 (M-PCR 1) identified five types of ccr genes; M-PCR 2 identified class A to class C mec ; M-PCRs 3 and 4 identified specific open reading frames in the J1 regions of type I and IV and of type II, III, and V SCC mec elements, respectively; M-PCR 5 identified the transposons Tn 554 and ΨTn 554 integrated into the J2 regions of type II and III SCC mec elements; and M-PCR 6 identified plasmids pT181 and pUB110 integrated into J3 regions. The system was validated with 99 MRSA strains carrying SCC mec elements of different types. The SCC mec types of 93 out of the 99 MRSA strains could be assigned. The SCC mec type assignments were identical to those made with a PCR system that uses numerous primer pairs to identify genes or gene alleles. Our system of six M-PCRs is thus a convenient and reliable method for typing SCC mec elements.

Staphylococcus haemolyticus is an opportunistic bacterial pathogen that colonizes human skin and is remarkable for its highly antibiotic-resistant phenotype. We determined the complete genome sequence of S.haemolyticus to better understand its pathogenicity and evolutionary relatedness to the other staphylococcal species. A large proportion of the open reading frames in the genomes of S.haemolyticus , Staphylococcus aureus , and Staphylococcus epidermidis were conserved in their sequence and order on the chromosome. We identified a region of the bacterial chromosome just downstream of the origin of replication that showed little homology among the species but was conserved among strains within a species. This novel region, designated the “ oriC environ,” likely contributes to the evolution and differentiation of the staphylococcal species, since it was enriched for species-specific nonessential genes that contribute to the biological features of each staphylococcal species. A comparative analysis of the genomes of S.haemolyticus , S.aureus , and S.epidermidis elucidated differences in their biological and genetic characteristics and pathogenic potentials. We identified as many as 82 insertion sequences in the S.haemolyticus chromosome that probably mediated frequent genomic rearrangements, resulting in phenotypic diversification of the strain. Such rearrangements could have brought genomic plasticity to this species and contributed to its acquisition of antibiotic resistance.

Oxygen depletion of Mycobacterium tuberculosis engages the DosR regulon that coordinates an overall down-regulation of metabolism while up-regulating specific genes involved in respiration and central metabolism. We have developed a chemostat model of M. tuberculosis where growth rate was a function of dissolved oxygen concentration to analyze metabolic adaptation to hypoxia. A drop in dissolved oxygen concentration from 50 mmHg to 0.42 mmHg led to a 2.3 fold decrease in intracellular ATP levels with an almost 70-fold increase in the ratio of NADH/NAD⁺. This suggests that re-oxidation of this co-factor becomes limiting in the absence of a terminal electron acceptor. Upon oxygen limitation genes involved in the reverse TCA cycle were upregulated and this upregulation was associated with a significant accumulation of succinate in the extracellular milieu. We confirmed that this succinate was produced by a reversal of the TCA cycle towards the non-oxidative direction with net CO₂ incorporation by analysis of the isotopomers of secreted succinate after feeding stable isotope (¹³C) labeled precursors. This showed that the resulting succinate retained both carbons lost during oxidative operation of the TCA cycle. Metabolomic analyses of all glycolytic and TCA cycle intermediates from ¹³C-glucose fed cells under aerobic and anaerobic conditions showed a clear reversal of isotope labeling patterns accompanying the switch from normoxic to anoxic conditions. M. tuberculosis encodes three potential succinate-producing enzymes including a canonical fumarate reductase which was highly upregulated under hypoxia. Knockout of frd, however, failed to reduce succinate accumulation and gene expression studies revealed a compensatory upregulation of two homologous enzymes. These major realignments of central metabolism are consistent with a model of oxygen-induced stasis in which an energized membrane is maintained by coupling the reductive branch of the TCA cycle to succinate secretion. This fermentative process may offer unique targets for the treatment of latent tuberculosis. Tuberculosis in its latent form infects one-third of the total human population, hiding in structures called granulomas in the lung. The dense tissue formed by the granuloma severely limits the amount of oxygen available and yet somehow the bacteria manage to survive for many years. In this study we have examined TB bacteria artificially locked at specific oxygen tensions to understand how they maintain basic metabolic functions in the absence of oxygen. As oxygen is lowered intracellular ATP levels fall and reduced cofactors such as NADH accumulate, unable to close the respiratory cycle using electron transport to donate their extra electrons to molecular oxygen. As they begin to suffocate, the bacteria flip the direction of their tricarboxylic acid (TCA) cycle enzymes from an oxidative direction to a reductive direction and begin to actively secrete succinic acid. Similar to lactate secretion in bacteria capable of growing anaerobically, this process is fermentative in nature, sufficient to enable the TB bacteria to continue basic physiologic functions like maintaining a proton gradient across the membrane, but not sufficient to allow them to grow. Understanding these processes may pinpoint vulnerabilities that could help develop interventions to treat latent infections before people develop destructive lung disease.

Phage therapy, the use of bacteriophages (phages) to treat bacterial infections, is regaining momentum as a promising weapon against the rising threat of multidrug-resistant (MDR) bacteria. This comprehensive review explores the historical context, the modern resurgence of phage therapy, and phage-facilitated advancements in medical and technological fields. It details the mechanisms of action and applications of phages in treating MDR bacterial infections, particularly those associated with biofilms and intracellular pathogens. The review further highlights innovative uses of phages in vaccine development, cancer therapy, and as gene delivery vectors. Despite its targeted and efficient approach, phage therapy faces challenges related to phage stability, immune response, and regulatory approval. By examining these areas in detail, this review underscores the immense potential and remaining hurdles in integrating phage-based therapies into modern medical practices.

The production of staphylocoagulase (SC) causing the plasma coagulation is one of the important characteristics of Staphylococcus aureus. Although SCs have been classified into 10 serotypes based on the differences in the antigenicity, genetic bases for their diversities and relatedness to chromosome types are poorly understood. We compared the nucleotide sequences of 105 SC genes (coa), 59 of which were determined in this study. D1 regions, which contain prothrombin-activating and -binding domains and are presumed to be the binding site of each type-specific antiserum, were classified into twelve clusters having more than 90% nucleotide identities, resulting to create two novel SC types, XI and XII, in addition to extant 10 types. Nine of the twelve SC types were further subdivided into subtypes based on the differences of the D2 or the central regions. The phylogenetical relations of the D1 regions did not correlate exactly with either one of agr types and multilocus sequence types (STs). In addition, genetic analysis showed that recombination events have occurred in and around coa. So far tested, STs of 126 S. aureus strains correspond to the combination of SC type and agr type except for the cases of CC1 and CC8, which contained two and three different SC types, respectively. The data suggested that the evolution of coa was not monophyletic in the species. Chromosomal recombination had occurred at coa and agr loci, resulting in the carriage of the combinations of allotypically different important virulence determinants in staphylococcal chromosome.