Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most prevalent multidrug-resistant pathogens worldwide, exhibiting increasing resistance to the latest antibiotic therapies. Here we show that the triple β-lactam combination meropenem/piperacillin/tazobactam (ME/PI/TZ) acts synergistically and is bactericidal against MRSA N315 and 72 clinical MRSA isolates in vitro, and clears MRSA N315 infection in a mouse model. ME/PI/TZ suppresses evolution of resistance in MRSA via reciprocal collateral sensitivity of its constituents. We demonstrate that these activities also extend to other carbapenem/penicillin/β-lactamase inhibitor combinations. ME/PI/TZ circumvents the tight regulation of the mec and bla operons in MRSA, the basis for inducible resistance to β-lactam antibiotics. Furthermore, ME/PI/TZ subverts the function of penicillin-binding protein 2a (PBP2a) action via allostery, which we propose as the mechanism for both synergy and collateral sensitivity. Showing similar in vivo activity to linezolid, ME/PI/TZ demonstrates that combinations of older β-lactam antibiotics could be effective against MRSA infections in humans.

Osteomyelitis (OM), or inflammation of bone tissue, occurs most frequently as a result of bacterial infection and severely perturbs bone structure. OM is predominantly caused by Staphylococcus aureus, and even with proper treatment, OM has a high rate of recurrence and chronicity. While S. aureus has been shown to infect osteoblasts, it remains unclear whether osteoclasts (OCs) are also a target of intracellular infection. Here, we demonstrate the ability of S. aureus to intracellularly infect and divide within OCs. OCs were differentiated from bone marrow macrophages (BMMs) by exposure to receptor activator of nuclear factor kappa-B ligand (RANKL). By utilizing an intracellular survival assay and flow cytometry, we found that at 18 h postinfection the intracellular burden of S. aureus increased dramatically in cells with at least 2 days of RANKL exposure, while the bacterial burden decreased in BMMs. To further explore the signals downstream of RANKL, we manipulated factors controlling OC differentiation, NFATc1 and alternative NF-κB, and found that intracellular bacterial growth correlates with NFATc1 levels in RANKL-treated cells. Confocal and time-lapse microscopy in mature OCs showed a range of intracellular infection that correlated inversely with S. aureus-phagolysosome colocalization. The propensity of OCs to become infected, paired with their diminished bactericidal capacity compared to BMMs, could promote OM progression by allowing S. aureus to evade initial immune regulation and proliferate at the periphery of lesions where OCs are most abundant. IMPORTANCE The inflammation of bone tissue is called osteomyelitis, and most cases are caused by an infection with the bacterium Staphylococcus aureus. To date, the bone-building cells, osteoblasts, have been implicated in the progression of these infections, but not much is known about how the bone-resorbing cells, osteoclasts, participate. In this study, we show that S. aureus can infect osteoclasts and proliferate inside these cells, whereas bone-residing macrophages, immune cells related to osteoclasts, destroy the bacteria. These findings elucidate a unique role for osteoclasts to harbor bacteria during infection, providing a possible mechanism by which bacteria could evade destruction by the immune system.

1. The b-type haem centres of the three (a, fi and y) subunit nitrate reductase from Paracoccus denitrijkans have been analysed by redox potentiometry. Two components were identified with mid-point potentials + 95 mV and +210 mV.2. Washing, in the absence of Mg2+ ions, of cytoplasmic membrane vesicles from P. denitriJicans promoted selective release of nitrate reductase activity. The released enzyme was purified by chromatography and shown to contain a and fi, but not y polypeptides. A haem spectrum was absent, consistent with the lack of the y subunit. The CI and fi polypeptides of the water-soluble nitrate reductase had molecular masses that were identical to those of the detergent-purified enzyme and also of the nitrate reductase in cytoplasmic membranes. This observation, together with the failure of protease inhibitors to prevent release from the membrane, indicates that the release is not related to limited proteolysis of the a and/or polypeptides. The relative molecular mass of the watersoluble afi enzyme was estimated to be approximately 200 000.3. The water-soluble nitrate reductase was released from intact inverted cytoplasmic membrane vesicles as jugded by loss of NADH-NO; reductase activity and retention by the vesicles after washing of uncoupler-sensitive NADH-oxidase activity. These observations show that a and fi polypeptides, and therefore the active site for nitrate reduction, are located on the cytoplasmic side of the membrane.4. Attempts to reverse the nitrate reductase activity of the enzyme, using nitrite as reductant plus ferricyanide or chlorate as tested oxidants, were unsuccessful. The implications for the mechanism of the enzyme are discussed.The respiratory nitrate reductase catalyses the first step of bacterial denitrification. Despite this important role, relatively little is known about these enzymes from denitrifying organisms, although there has been fairly extensive study of the nitrate reductase from Escherichia coli, an organism which does not denitrify [l, 21. We recently showed that for the denitrifier Paracoccus denitrzjicans the nitrate reductase can be purified in the presence of detergent from cytoplasmic membranes in a form that has three polypeptides, designated a, p and y. These have similar molecular masses to the corresponding components of the E. coli enzyme [3]. A further similarity between the enzymes is that the y subunit has in each case been identified as the cytochrome subunit [l -31.Hackett and Bragg [4] have presented evidence that a partially purified nitrate reductase from E. coli contains two b-type haem centres that can be distinguished on the basis of redox potential. Such observations, together with the evidence that the b-type cytochrome of the bcl complex of mitochondria and photosynthetic bacteria contains two haem groups [51, prompted analysis, reported here, of this aspect of the purified nitrate reductase from P. denitrijkans.A pre-requisite for understanding the functioning of the nitrate reductase in P. denitrificans is the determination of the...

Normal and rearranged RNA segments 10 of group A rotaviruses isolated from a chronically infected immunodeficient child were amplified by the polymerase chain reaction as full-length cDNA copies, and were subsequently cloned and sequenced. Compared with the nucleotide sequence of the normal RNA segment 10, the rearranged form contains a partial non-coding duplication at its 3' end and several point mutations. The normal RNA segment 10 was similar to that of bovine rotavirus.

A real-time PCR hybridization assay for Legionella pneumophila is described; the assay uses LightCycler (Idaho Technology) methodology to specifically detect 2.5 CFU/reaction, equivalent to 1,000 CFU/liter of starting water sample. The assay, including DNA extraction and confirmation of product identity, is completed within 90 min of receipt of a sample.

No abstract

Steroid 21-hydroxylase deficiency is the leading cause of impaired cortisol synthesis in congenital adrenal hyperplasia (CAH), with the nonclassic form (NC) comprising approximately 1% of the Caucasian population. The structure of the CYP21 gene was studied in 13 unrelated NC-CAH patients, three affected siblings, and 55 blood donors using polymerase chain reaction. In addition to the Leu-281 and Leu-30 mutations previously associated with NC-CAH, the finding of a Pro-453 to Ser mutation in exon-10 of CYP21 in the NC-CAH patients is reported. Ser-453 was found in 46.2% of unrelated NC-CAH patients, but only 7.7% and 3.6% of salt-wasting CAH patients and blood donors, respectively. In contrast to the Leu-281 and Leu-30 mutations, Ser-453 has not been previously detected in the CYP21 pseudogene (CYP21P) and, therefore, has not likely arisen by gene conversion.

e Using serial-section transmission electron microscopy and three-dimensional (3D) electron tomography, we characterized membrane dynamics that accompany the construction of a nuclear exchange junction between mating cells in the ciliate Tetrahymena thermophila. Our methods revealed a number of previously unknown features. (i) Membrane fusion is initiated by the extension of hundreds of 50-nm-diameter protrusions from the plasma membrane. These protrusions extend from both mating cells across the intercellular space to fuse with membrane of the mating partner. (ii) During this process, small membranebound vesicles or tubules are shed from the plasma membrane and into the extracellular space within the junction. The resultant vesicle-filled pockets within the extracellular space are referred to as junction lumens. (iii) As junction lumens fill with extracellular microvesicles and swell, the plasma membrane limiting these swellings undergoes another deformation, pinching off vesicle-filled vacuoles into the cytoplasm (reclamation). (iv) These structures (resembling multivesicular bodies) seem to associate with autophagosomes abundant near the exchange junction. We propose a model characterizing the membrane-remodeling events that establish cytoplasmic continuity between mating Tetrahymena cells. We also discuss the possible role of nonvesicular lipid transport in conditioning the exchange junction lipid environment. Finally, we raise the possibility of an intercellular signaling mechanism involving microvesicle shedding and uptake.