Methicillin-resistant Staphylococcus aureus (MRSA) strains have become prevalent in health care facilities and in the community worldwide (3, 4). MRSA strains produce penicillin binding protein 2Ј or 2a, which is poorly acylated by ␤-lactam antibiotics (5,22,25). The mecA gene, encoding PBP2a, is carried on a peculiar type of mobile genetic element inserted into the staphylococcal chromosome, designated staphylococcal cassette chromosome mec (SCCmec) elements (12,14,24).SCCmec elements typically share four characteristics: first, they carry the mec gene complex (mec) consisting of the methicillin resistance determinant mecA and its regulatory genes and insertion sequences; second, they carry the ccr gene complex (ccr) consisting of ccr genes that are responsible for the mobility of the element and its surrounding sequences; third, they have characteristic directly repeated nucleotide sequences and inverted complementary sequences at both ends; and last, they integrate into the 3Ј end of an open reading frame (ORF), orfX.Despite these similarities, the structures of SCCmec elements are rather divergent. Allotypic differences that are used for SCCmec type definitions have been identified in both ccr and mec. Five types of ccr and four classes of mec have been reported. ccr types 1 to 4 carry the ccrA and ccrB genes, which share approximately 80% identity with each other, and the type 5 ccr carries the ccrC gene (10,11,17,19). Four classes of the mec gene complexes have been identified among methicillinresistant staphylococcal strains of various species: class A mec, consisting of IS431mec-mecA-mecR1-mecI; class B mec, consisting of IS431mec-mecA-⌬mecR1-IS1272; class C mec, consisting of IS431mec-mecA-⌬mecR1-IS431; and class D mec, consisting of IS431mec-mecA-⌬mecR1 with no insertion sequences downstream of ⌬mecR1 identified by PCR as of yet (13). In S. aureus strains, mec classes A, B, and C have been identified. Insertion sequences have sometimes been found to be integrated in or around the class A mec. A class A mec carrying IS431 downstream of mecI was found in Staphylococcus haemolyticus (13). Recently, Shore et al. identified MRSA strains carrying class A mec with an insertion of IS1182 in and around the mecI gene and designated them classes A3 and A4 (23).The SCCmec element type has been defined by the combination of ccr type and mec class. In MRSA strains, six types of SCCmec elements, that is, six combinations of ccr and mec, have been reported (Table 1). These six SCCmec elements have been further classified by differences in regions other than ccr and mec, which are designated junkyard (J) regions. The J regions comprise three parts: J1 (the region between ccr and the right-flanking chromosomal region), J2 (the region between mec and ccr), and J3 (the region between orfX and mec). The J regions are not always specific to each SCCmec type, but certain J regions are commonly shared among certain types of SCCmec elements. Of the three regions, we regard J1 as being the most fundamental, because we presume t...

A simple and reliable method using a polymerase chain reaction (PCR) was devised to identify methicillinresistant staphylococci. By using lysates of the strain to be tested as templates and 22-mer oligonucleotides as primers, a 533-bp region of mecA, the structural gene of a low-affinity penicillin-binding protein (PBP 2'), was amplified by PCR and detected by agarose gel electrophoresis. Results obtained by this method were compared with those obtained by broth microdilution MIC determination for 210 and 100 clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci, respectively. Of 99 mecA-negative S. aureus isolates, 100% of the strains were methicillin susceptible and 98% of the strains were oxacillin susceptible. Three strains (3%) of 111 mecA-positive S. aureus isolates exhibited almost the same susceptibility to ,(-lactams as the mecA-negative ones and did not produce detectable amounts of PBP 2' despite the presence of the mecA gene. One of them yielded typically methicillin-resistant variants at a low frequency with concomitant recovery of PBP 2' production. The mecA gene was also found in coagulase-negative Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus sciuri, Staphylococcus saprophyticus, and Staphylococcus caprae and conferred resistance on most of the bacteria. * Corresponding author. species were identified by Staphyogram (Terumo, Tokyo, Japan), an identification kit, and the coagulase test. S. aureus SR3626, SR3633, SR3636, SR3639, SR3665, SR3681, and SR3716 were used as mecA-positive control organisms (12), and S. aureus ATCC 25923, 209P JC-1, and Smith were used as mecA-negative control organisms.

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.As a part of the normal bacterial flora, staphylococci colonize the skin and mucosal membranes of humans. In addition, staphylococci frequently cause opportunistic infections in patients with underlying disease, such as those with prosthetic devices, surgical patients, individuals undergoing dialysis, or patients with diabetes. Since antibiotic chemotherapy was introduced in the last century, staphylococci have successfully persisted by altering their genetic traits to avoid being killed. Multidrug-resistant staphylococcal strains, exemplified by methicillin-resistant staphylococci, are now prevalent worldwide. Among 40 staphylococcal species described to date, Staphylococcus aureus is the most virulent species and poses the greatest threat in hospitals worldwide. In addition to its nosocomial spread, S. aureus has also become problematic in community settings, where individuals without predisposing factors have acquired methicillin-resistant staphylococcal infections. Although most community-acquired staphylococcal infections involve the skin and soft tissues, some otherwise healthy children have acquired potentially lethal S. aureus infections with severe symptoms, such as necrotizing pneumonia (5,28,29).To gain a better understanding of the overall pathogenesis of staphylococcal infections and identify novel targets for new chemotherapeutic agents, researchers have sequenced the genomes of seven S. aureus strains (3,9,13,22). A comparative analysis of these strains has revealed that many genes involved in staphylococcal pathogenicity and...

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.

We have identified a cytomegalovirus virion protein capable of modulating the rapid induction of an interferon-like response in cells that follows virus binding and penetration. Functional genomics revealed a role for the major cytomegalovirus structural protein, pp65 (ppUL83), in counteracting this response. The underlying mechanism involves a differential impact of this structural protein on the regulation of interferon response factor 3 (IRF-3). In contrast, NF-B is activated independent of pp65, and neither STAT1 nor STAT3 becomes activated by either virus. pp65 is sufficient to prevent the activation of IRF-3 when introduced alone into cells. pp65 acts by inhibiting nuclear accumulation of IRF-3 and is associated with a reduced IRF-3 phosphorylation state. Thus, this investigation shows that the major structural protein of cytomegalovirus is committed to the modulation of the IRF-3 response, a primary mediator of the type I interferon response. By subverting IRF-3, the virus escapes throwing a central alarm devoted to both immediate antiviral control and regulation of the immune response.

bEndogenous retroviruses (ERVs) comprise a significant percentage of the mammalian genome, and it is poorly understood whether they will remain as inactive genomes or emerge as infectious retroviruses. Although several types of ERVs are present in domestic cats, infectious ERVs have not been demonstrated. Here, we report a previously uncharacterized class of endogenous gammaretroviruses, termed ERV-DCs, that is present and hereditary in the domestic cat genome. We have characterized a subset of ERV-DC proviral clones, which are numbered according to their genomic insertions. One of these, ERV-DC10, located in the q12-q21 region on chromosome C1, is an infectious gammaretrovirus capable of infecting a broad range of cells, including human. Our studies indicate that ERV-DC10 entered the genome of domestic cats in the recent past and appeared to translocate to or reintegrate at a distinct locus as infectious ERV-DC18. Insertional polymorphism analysis revealed that 92 of 244 domestic cats had ERV-DC10 on a homozygous or heterozygous locus. ERV-DC-like sequences were found in primate and rodent genomes, suggesting that these ERVs, and recombinant viruses such as RD-114 and BaEV, originated from an ancestor of ERV-DC. We also found that a novel recombinant virus, feline leukemia virus subgroup D (FeLV-D), was generated by ERV-DC env transduction into feline leukemia virus in domestic cats. Our results indicate that ERV-DCs behave as donors and/or acceptors in the generation of infectious, recombinant viruses. The presence of such infectious endogenous retroviruses, which could be harmful or beneficial to the host, may affect veterinary medicine and public health.

The emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. Here we report the development of a series of CRISPR-Cas13a-based antibacterial nucleocapsids, termed Cap-sidCas13a(s), capable of sequence-specific killing of carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus by recognizing corresponding antimicrobial resistance genes. CapsidCas13a constructs are generated by packaging programmed CRISPR-Cas13a into a bacteriophage capsid to target antimicrobial resistance genes. Contrary to Cas9-based antimicrobials that lack bacterial killing capacity when the target genes are located on a plasmid, the CapsidCas13a(s) exhibit strong bacterial killing activities upon recognizing target genes regardless of their location. Moreover, we also demonstrate that the CapsidCas13a(s) can be applied to detect bacterial genes through gene-specific depletion of bacteria without employing nucleic acid manipulation and optical visualization devices. Our data underscore the potential of CapsidCas13a(s) as both therapeutic agents against antimicrobial-resistant bacteria and nonchemical agents for detection of bacterial genes.

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