Search

The article discusses the growing threat of multidrug-resistant Gram-negative infections. Topics discussed include the rapid evolution of bacterial resistance mechanisms, the pharmaceutical industry's challenges in developing effective antibiotics, and a poignant case study of fatal antibiotic-resistant E. coli in a child.

Acinetobacter baumannii is a Gram-negative organism listed as an urgent threat pathogen by the World Health Organization (WHO). Carbapenem-resistant A. baumannii (CRAB), especially, present therapeutic challenges due to complex mechanisms of resistance to β-lactams. One of the most important mechanisms is the production of β-lactamase enzymes capable of hydrolyzing β-lactam antibiotics. Co-expression of multiple classes of β-lactamases is present in CRAB; therefore, the design and synthesis of "cross-class" inhibitors is an important strategy to preserve the efficacy of currently available antibiotics. To identify new, nonclassical β-lactamase inhibitors, we previously identified a sulfonamidomethaneboronic acid CR167 active against Acinetobacter-derived class C β-lactamases (ADC-7). The compound demonstrated affinity for ADC-7 with a Ki = 160 nM and proved to be able to decrease MIC values of ceftazidime and cefotaxime in different bacterial strains. Herein, we describe the activity of CR167 against other β-lactamases in A. baumannii: the cefepime-hydrolysing class C extended-spectrum β-lactamase (ESAC) ADC-33 and the carbapenem-hydrolyzing OXA-24/40 (class D). These investigations demonstrate CR167 as a valuable cross-class (C and D) inhibitor, and the paper describes our attempts to further improve its activity. Five chiral analogues of CR167 were rationally designed and synthesized. The structures of OXA-24/40 and ADC-33 in complex with CR167 and select chiral analogues were obtained. The structure activity relationships (SARs) are highlighted, offering insights into the main determinants for cross-class C/D inhibitors and impetus for novel drug design. [ABSTRACT FROM AUTHOR]

The recent emergence of multidrug resistance (MDR) in Acinetobacter baumannii has raised concern in health care settings worldwide. In order to understand the repertoire of resistance determinants and their organization and origins, we compared the genome sequences of three MDR and three drug-susceptible A. baumannii isolates. The entire MDR phenotype can be explained by the acquisition of discrete resistance determinants distributed throughout the genome. A comparison of closely related MDR and drug-susceptible isolates suggests that drug efflux may be a less significant contributor to resistance to certain classes of antibiotics than inactivation enzymes are. A resistance island with a variable composition of resistance determinants interspersed with transposons, integrons, and other mobile genetic elements is a significant but not universal contributor to the MDR phenotype. Four hundred seventy-five genes are shared among all six clinical isolates but absent from the related environmental species Acinetobacter baylyi ADP1. These genes are enriched for transcription factors and transporters and suggest physiological features of A. baumannii that are related to adaptation for growth in association with humans.

The [Ca.sup.2+]-sensing receptor (CAR), a G protein-coupled receptor, is expressed in many epithelial tissues including the parathyroid glands, kidney, and GI tract. Although its role in regulating PTH levels and [Ca.sup.2+] metabolism are best characterized, it may also regulate salt and water transport in the kidney as demonstrated by recent reports showing association of potent gain-of-function mutations in the CaR with a Bartter-like, salt-wasting phenotype. To determine whether this receptor interacts with novel proteins that control ion transport, we screened a human adult kidney cDNA library with the COOH-terminal 219 amino acid cytoplasmic tail of the CaR as bait using the yeast two-hybrid system. We identified two independent clones coding for ~125 aa from the COOH terminus of the inwardly rectifying [K.sup.+] channel, Kir4.2. The CaR and Kir4.2 as well as Kir4.1 (another member of Kir4 subfamily) were reciprocally coimmunoprecipitated from HEK-293 cells in which they were expressed, but the receptor did not coimmunoprecipitate with Kir5.1 or Kir1.1. Both Kir4.1 and Kir4.2 were immunoprecipitated from rat kidney extracts with the CaR. In Xenopus laevis oocytes, expression of the CaR with either Kir4.1 or Kir4.2 channels resulted in inactivation of whole cell current as measured by two-electrode voltage clamp, but the nonfunctional CaR mutant Ca[R.sup.R796W], and that does not coimmunoprecipitate with the channels, had no effect. Kir4.1 and the CaR were colocalized in the basolateral membrane of the distal nephron. The CaR interacts directly with Kir4.1 and Kir4.2 and can decrease their currents, which in turn could reduce recycling of [K.sup.+] for the basolateral [Na.sup.+]-[K.sup.+]-ATPase and thereby contribute to inhibition of [Na.sup.+] reabsorption. inwardly rectifying [K.sup.+] channel; HEK 293 cells; yeast two hybrid

The [Ca.sup.2+]-sensing receptor (CAR) couples to multiple G proteins involved in distinct signaling pathways: [G[alpha].sub.i] to inhibit the activity of adenylyl cyclase and activate ERK, [G[alpha].sub.q] to stimulate phospholipase C and phospholipase [A.sub.2], and G[beta][gamma] to stimulate phosphatidylinositol 3-kinase. To determine whether the receptor also couples to [G[alpha].sub.12/13], we investigated the signaling pathway by which the CaR regulates phospholipase D (PLD), a known [G[alpha].sub.12/13] target. We established Madin-Darby canine kidney (MDCK) cell lines that stably overexpress the wild-type CaR (Ca[R.sup.WT]) or the nonfunctional mutant Ca[R.sup.R796W] as a negative control, prelabeled these cells with [[sup.3]H]palmitic acid, and measured CaR-stimulated PLD activity as the formation of [[sup.3]H]phosphatidylethanol (PEt). The formation of [[sup.3]H]PEt increased in a time-dependent manner in the cells that overexpress the Ca[R.sup.WT] but not the Ca[R.sup.R796W]. Treatment of the cells with [C.sub.3] exoenzyme inhibited PLD activity, which indicates that the CaR activates the Rho family of small G proteins, targets of [G[alpha].sub.12/13]. To determine which G protein(s) the CaR couples to in order to activate Rho and PLD, we pretreated the cells with pertussis toxin to inactivate [G[alpha].sub.i] or coexpressed regulators of G protein-signaling (RGS) proteins to attenuate G protein signaling (RGS4 for [G[alpha].sub.i] and [G[alpha].sub.q], and a p115RhoGEF construct containing the RGS domain for [G[alpha].sub.12/13]). Overexpression of p115RhoGEF-RGS in the MDCK cells that overexpress Ca[R.sup.WT] inhibited extracellular [Ca.sup.2+]-stimulated PLD activity, but pretreatment of cells with pertussis toxin and overexpression of RGS4 were without effect. The involvement of other signaling components such as protein kinase C, ADP-ribosylation factor, and phosphatidylinositol biphosphate was excluded. These findings demonstrate that the CaR couples to [G[alpha].sub.12/13] to regulate PLD via a Rho-dependent mechanism and does so independently of [G[alpha].sub.i] and [G[alpha].sub.q]. This suggests that the CaR may regulate cytoskeleton via [G[alpha].sub.12/13], Rho, and PLD. calcium-sensing receptor; G proteins; RGS proteins

Mutagenesis studies were performed to better understand the basis of the inhibitor-resistant phenotype in SHV A [beta]-lactamase and also examine the role of a second-shell residue, Asn276. The results confirmed residue 276 as an important second-shell residue in class A [beta]-lactamase-mediated resistance to substrates and inhibitors, and also revealed Asn ablility to precisely modulate the conformational flexibility of Arg244 required for successful evolution in nature.

Background Overcoming β-lactam resistance in pathogens such as Pseudomonas aeruginosa is a major clinical challenge. Rapid molecular diagnostics (RMDs) have the potential to inform selection of empiric therapy in patients infected by P. aeruginosa. Methods In this study, we used a heterogeneous collection of 197 P. aeruginosa that included multidrug-resistant isolates to determine whether 2 representative RMDs (Acuitas Resistome test and VERIGENE gram-negative blood culture test) could identify susceptibility to 2 newer β-lactam/β-lactamase inhibitor (BL-BLI) combinations, ceftazidime/avibactam (CZA) and ceftolozane/tazobactam (TOL/TAZO). Results We found that the studied RMD platforms were able to correctly identify BL-BLI susceptibility (susceptibility sensitivity, 100%; 95% confidence interval [CI], 97%, 100%) for both BLs-BLIs. However, their ability to detect resistance to these BLs-BLIs was lower (resistance sensitivity, 66%; 95% CI, 52%, 78% for TOL/TAZO and 33%; 95% CI, 20%, 49% for CZA). Conclusions The diagnostic platforms studied showed the most potential in scenarios where a resistance gene was detected or in scenarios where a resistance gene was not detected and the prevalence of resistance to TOL/TAZO or CZA is known to be low. Clinicians need to be mindful of the benefits and risks that result from empiric treatment decisions that are based on resistance gene detection in P. aeruginosa, acknowledging that such decisions are impacted by the prevalence of resistance, which varies temporally and geographically. [ABSTRACT FROM AUTHOR]

Background. Polymyxins including colistin are an important "last-line" treatment for infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKp). Increasing use of colistin has led to resistance to this cationic antimicrobial peptide. Methods. A cohort nested within the Consortium on Resistance against Carbapenems in Klebsiella pneumoniae (CRACKLE) was constructed of patients with infection, or colonization with CRKp isolates tested for colistin susceptibility during the study period of December, 2011 to October, 2014. Reference colistin resistance determination as performed by broth macrodilution was compared to results from clinical microbiology laboratories (Etest) and to polymyxin resistance testing. Each patient was included once, at the time of their first colistin-tested CRKp positive culture. Time to 30-day in-hospital all-cause mortality was evaluated by Kaplan-Meier curves and Cox proportional hazard modeling. Results. In 246 patients with CRKp, 13% possessed ColR CRKp. ColR was underestimated by Etest (very major error rate = 35%, major error rate = 0.4%). A variety of rep-PCR strain types were encountered in both the ColS and the ColR groups. Carbapenem resistance was mediated primarily by blaKPC-2 (46%) and blaKPC-3 (50%). ColR was associated with increased hazard for in-hospital mortality (aHR 3.48; 95% confidence interval, 1.73-6.57; P < .001). The plasmid-associated ColR genes, mcr-1 and mcr-2 were not detected in any of the ColR CRKp. Conclusions. In this cohort, 13% of patients with CRKp presented with ColR CRKp. The apparent polyclonal nature of the isolates suggests de novo emergence of ColR in this cohort as the primary factor driving ColR. Importantly, mortality was increased in patients with ColR isolates. [ABSTRACT FROM AUTHOR]

The medical community needs systematic and pragmatic approaches for evaluating the benefit-risk trade-offs of diagnostics that assist in medical decision making. Benefit-Risk Evaluation of Diagnostics: A Framework (BED-FRAME) is a strategy for pragmatic evaluation of diagnostics designed to supplement traditional approaches. BED-FRAME evaluates diagnostic yield and addresses 2 key issues: (1) that diagnostic yield depends on prevalence, and (2) that different diagnostic errors carry different clinical consequences. As such, evaluating and comparing diagnostics depends on prevalence and the relative importance of potential errors. BED-FRAME provides a tool for communicating the expected clinical impact of diagnostic application and the expected tradeoffs of diagnostic alternatives. BED-FRAME is a useful fundamental supplement to the standard analysis of diagnostic studies that will aid in clinical decision making. [ABSTRACT FROM AUTHOR]

Background. Successful treatment of infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa is thwarted by the emergence of antibiotic resistance and biofilm formation on prosthetic devices. Our aims were to decipher the molecular basis of resistance in a unique case of prosthetic valve endocarditis (PVE) caused by MDR P. aeruginosa. Methods. Five sequential MDR P. aeruginosa blood isolates collected during a 7-month period were recovered from a patient suffering from PVE previously exposed to β-lactam antibiotics. Minimum inhibitory concentrations (MICs) of several classes of antibiotics were used to indicate clinical resistance characteristics; relatedness of the isolates was determined using multilocus sequence typing and repetitive sequence-based polymerase chain reaction. Amplification and sequencing of regulatory and resistance genes was performed. Results. All isolates belonged to ST 298, possessed blaPDC-16, and were resistant to fluoroquinolones and carbapenems. In the course of therapy, we observed a >2-fold increase in cephalosporin resistance (4 μg/mL to >16 μg/mL). Sequencing of the AmpC regulator, ampR, revealed a D135N point mutation in cephalosporin-resistant isolates. Common carbapenemase genes were not identified. All isolates demonstrated a premature stop codon at amino acid 79 of the outer membrane protein OprD and mutations in the quinolone resistance-determining regions of gyrA and parC. Point mutations in nalC, an efflux pump regulator, were also observed. Conclusions. In this analysis, we chart the molecular evolution of β-lactam resistance in a case of PVE.We show that mutations in regulatory genes controlling efflux and cephalosporinase production contributed to the MDR phenotype. [ABSTRACT FROM AUTHOR]

Background: Rapid molecular diagnostic (RMD) platforms may lead to better antibiotic use. Our objective was to develop analytical strategies to enhance the interpretation of RMDs for clinicians. Methods: We compared the performance characteristics of 4 RMD platforms for detecting resistance against β-lactams in 72 highly resistant isolates of Escherichia coli and Klebsiella pneumoniae (PRIMERS I). Subsequently, 2 platforms were used in a blinded study in which a heterogeneous collection of 196 isolates of E. coli and K. pneumoniae (PRIMERS II) were examined. We evaluated the genotypic results as predictors of resistance or susceptibility against β-lactam antibiotics. We designed analytical strategies and graphical representations of platform performance, including discrimination summary plots and susceptibility and resistance predictive values, that are readily interpretable by practitioners to inform decision-making. Results: In PRIMERS I, the 4 RMD platforms detected β-lactamase (bla) genes and identified susceptibility or resistance in >95% of cases. In PRIMERS II, the 2 platforms identified susceptibility against extended-spectrum cephalosporins and carbapenems in >90% of cases; however, against piperacillin/tazobactam, susceptibility was identified in <80% of cases. Applying the analytical strategies to a population with 15% prevalence of ceftazidime-resistance and 5% imipenem-resistance, RMD platforms predicted susceptibility in >95% of cases, while prediction of resistance was 69%-73% for ceftazidime and 41%-50% for imipenem. Conclusions: RMD platforms can help inform empiric β-lactam therapy in cases where bla genes are not detected and the prevalence of resistance is known. Our analysis is a first step in bridging the gap between RMDs and empiric treatment decisions. [ABSTRACT FROM AUTHOR]

Staphylococcus intermedius and Staphylococcus pseudintermedius are difficult to distinguish using conventional microbiological methods. Molecular diagnostic tools change our understanding of the epidemiology of these 2 organisms. In this study, we present (1) a detailed review of the current literature on molecular diagnostics and (2) a case series in which misidentification was proven in 1 case. We conclude that S pseudintermedius is a more common human pathogen than previously recognized. [ABSTRACT FROM AUTHOR]

Background. Klebsiella pneumoniae isolates harboring the K. pneumoniae carbapenemase gene (blaKPC) are creating a significant healthcare threat in both acute and long-term care facilities (LTCFs). As part of a study conducted in 2004 to determine the risk of stool colonization with extended-spectrum cephalosporin-resistant gram-negative bacteria, 12 isolates of K. pneumoniae that exhibited nonsusceptibility to extended-spectrum cephalosporins were detected. All were gastrointestinal carriage isolates that were not associated with infection. Methods. Reassessment of the carbapenem minimum inhibitory concentrations using revised 2011 Clinical Laboratory Standards Institute breakpoints uncovered carbapenem resistance. To further investigate, a DNA microarray assay, PCR-sequencing of bla genes, immunoblotting, repetitive-sequence-based PCR (rep-PCR) and multilocus sequence typing (MLST) were performed. Results. The DNA microarray detected blaKPC in all 12 isolates, and blaKPC-3 was identified by PCR amplification and sequencing of the amplicon. In addition, a blaSHV-11 gene was detected in all isolates. Immunoblotting revealed “low-level” production of the K. pneumoniae carbapenemase, and rep-PCR indicated that all blaKPC-3-positive K. pneumoniae strains were genetically related (≥98% similar). According to MLST, all isolates belonged to sequence type 36. This sequence type has not been previously linked with blaKPC carriage. Plasmids from 3 representative isolates readily transferred the blaKPC-3 to Escherichia coli J-53 recipients. Conclusions. Our findings reveal the “silent” dissemination of blaKPC-3 as part of Tn4401b on a mobile plasmid in Northeast Ohio nearly a decade ago and establish the first report, to our knowledge, of K. pneumoniae containing blaKPC-3 in an LTCF caring for neurologically impaired children and young adults. [ABSTRACT FROM AUTHOR]

Genotyping methods are essential to understand the transmission dynamics of Acinetobacter baumannii. We examined the representative genotypes of A. baumannii at different time periods in select locations in Ohio, using two rapid automated typing methods: PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS), a form of multi-locus sequence typing (MLST), and repetitive-sequence-based-PCR (rep-PCR). Our analysis included 122 isolates from 4 referral hospital systems, in 2 urban areas of Ohio. These isolates were associated with outbreaks at 3 different time periods (1996, 2000 and 2005-2007). Type assignments of PCR/ESI-MS and rep-PCR were compared to each other and to worldwide (WW) clone types. The discriminatory power of each method was determined using the Simpson's index of diversity (DI). We observed that PCR/ESI-MS sequence type (ST) 14, corresponding to WW clone 3, predominated in 1996, whereas ST 12 and 14 coexisted in the intermediate period (2000) and ST 10 and 12, belonging to WW clone 2, predominated more recently in 2007. The shift from WW clone 3 to WW clone 2 was accompanied by an increase in carbapenem resistance. The DI was approximately 0.74 for PCR/ESI-MS, 0.88 for rep-PCR and 0.90 for the combination of both typing methods. We conclude that combining rapid automated typing methods such as PCR/ESI-MS and rep-PCR serves to optimally characterize the regional molecular epidemiology of A. baumannii. Our data also sheds light on the changing sequence types in an 11 year period in Northeast Ohio. [ABSTRACT FROM AUTHOR]

Various combinations of antibiotics are reported to show synergy in treating nosocomial infections with multidrug-resistant ( MDR) Acinetobacter baumannii ( A. baumannii). Here, we studied hospital-acquired outbreak strains of MDR A. baumannii to evaluate optimal combinations of antibiotics. One hundred and twenty-one strains were grouped into one major and one minor clonal group based on repetitive PCR amplification. Twenty representative strains were tested for antibiotic synergy using Etest ®. Five strains were further analyzed by analytical isoelectric focusing and PCR to identify β-lactamase genes or other antibiotic resistance determinants. Our investigation showed that the outbreak strains of MDR A. baumannii belonged to two dominant clones. A combination of colistin and doxycycline showed the best result, being additive or synergistic against 70% of tested strains. Antibiotic additivity was observed more frequently than synergy. Strains possessing the same clonality did not necessarily demonstrate the same response to antibiotic combinations in vitro. We conclude that the effect of antibiotic combinations on our outbreak strains of MDR A. baumannii seemed strain-specific. The bacterial response to antibiotic combinations is probably a result of complex interactions between multiple concomitant antibiotic resistance determinants in each strain. [ABSTRACT FROM AUTHOR]

Objectives: New antibiotics that are active against multidrug-resistant (MDR) Acinetobacter baumannii are urgently needed. BAL30072, a siderophore monosulfactam antibiotic that rapidly penetrates the outer membrane of A. baumannii and has potent activity against most isolates, including those harbouring AmpC β-lactamases and metallo- (class B) or OXA- (class D) carbapenemases, is being developed to meet that need.Methods: We assessed the in vitro activity of BAL30072, meropenem and the combination of BAL30072 and meropenem (2:1 and 1:1 ratios) by MIC and time-kill studies. Proof-of-principle in vivo efficacy was determined using a rat soft-tissue infection model. Five diverse strains with defined phenotypic and genetic profiles were tested (AB307-0294, AB8407, AB1697, AB3340 and AB0057).Results: In microdilution assays, combining BAL30072 with meropenem lowered meropenem MICs 2-8-fold. In time-kill studies, the BAL30072 and meropenem combinations resulted in bactericidal concentrations 2-8-fold lower than those of meropenem or BAL30072 alone. In the rat model, BAL30072 was active against four of five strains (AB307-0294, AB8407, AB1697 and AB3340), including meropenem-susceptible and -non-susceptible strains. AB0057 was the only strain resistant to BAL30072 in vivo and in vitro (MIC >64 mg/L). Meropenem was active in vivo against two of the five strains tested (AB307-0294 and AB3340). Both BAL30072 and BAL30072 with meropenem were equally effective in vivo.Conclusions: These data support the continued evaluation of BAL30072 for use in the treatment of infections caused by MDR A. baumannii. [ABSTRACT FROM AUTHOR]

The Ca2+- sensing receptor (CaR), a G protein-coupled receptor, is expressed in many epithelial tissues including the parathyroid glands, kidney, and GI tract. Although its role in regulating PTH levels and Ca2+ metabolism are best characterized, it may also regulate salt and water transport in the kidney as demonstrated by recent reports showing association of potent gain-of-function mutations in the CaR with a Bartter-like, salt-wasting phenotype. To determine whether this receptor interacts with novel proteins that control ion transport, we screened a human adult kidney cDNA library with the COOH- terminal 219 amino acid cytoplasmic tail of the CaR as bait using the yeast two-hybrid system. We identified two independent clones coding for ~125 aa from the COOH terminus of the inwardly rectifying K+ channel, Kir4.2. The CaR and Kir4.2 as well as Kir4.1 (another member of Kir4 subfamily) were reciprocally coimmunoprecipitated from HEK-293 cells in which they were expressed, but the receptor did not coimmunoprecipitate with Kir5.1 or Kir1.1. Both Kir4.1 and Kir4.2 were immunoprecipitated from rat kidney extracts with the CaR. In Xenopus laevis oocytes, expression of the CaR with either Kir4.1 or Kir4.2 channels resulted in inactivation of whole cell current as measured by two-electrode voltage clamp, but the nonfunctional CaR mutant CaRR796W, and that does not coimmunoprecipitate with the channels, had no effect. Kir4.1 and the CaR were colocalized in the basolateral membrane of the distal nephron. The CaR interacts directly with Kir4.1 and Kir4.2 and can decrease their currents, which in turn could reduce recycling of K+ for the basolateral Na+-K+-ATPase and thereby contribute to inhibition of Na+ reabsorption. [ABSTRACT FROM AUTHOR]

Abstract: Filamin plays an important role in actin cytoskeleton organization, membrane stabilization, and anchoring of transmembrane proteins. Using short interfering RNA (siRNA) to selectively target the filamin A gene and silence its expression, we studied the role of filamin A in G protein coupled receptor (GPCR) signaling. Silencing of filamin A protein expression was determined by immunoblotting and immunofluorescence. Functional consequences of filamin A gene silencing were measured by studying its role in MAPK signaling pathways activated by the Ca2+-sensing receptor. This work defines filamin A involvement in GPCR signaling pathways and describes an additional method for studying its function. [Copyright &y& Elsevier]

The Ca[sup 2+]-sensing receptor (CaR) couples to multiple G proteins involved in distinct signaling pathways: Gα[sub i] to inhibit the activity of adenylyl cyclase and activate ERK, Gα[sub q] to stimulate phospholipase C and phospholipase A[sub 2], and Gβγ to stimulate phosphatidylinositol 3-kinase. To determine whether the receptor also couples to Gα[sub 12/13], we investigated the signaling pathway by which the CaR regulates phospholipase D (PLD), a known Gα[sub 12/13] target. We established Madin-Darby canine kidney (MDCK) cell lines that stably overexpress the wild-type CaR (CaR[sup WT]) or the nonfunctional mutant CaR[sup R796W] as a negative control, prelabeled these cells with [³H]palmitic acid, and measured CaRstimulated PLD activity as the formation of [³H]phosphatidylethanol (PEt). The formation of [³H]PEt increased in a time-dependent manner in the cells that overexpress the CaR[sup WT] but not the CaR[sup R796W]. Treatment of the cells with C[sub 3] exoenzyme inhibited PLD activity, which indicates that the CaR activates the Rho family of small G proteins, targets of Gα[sub l2/13]. To determine which G protein(s) the CaR couples to in order to activate Rho and PLD, we pretreated the cells with pertussis toxin to inactivate Gα[sub i] or coexpressed regulators of G protein-signaling (RGS) proteins to attenuate G protein signaling (RGS4 for Gα[sub i] and Gα[sub q], and a p115RhoGEF construct containing the RGS domain for Gα[sub 12/13]). Overexpression of p115RhoGEF-RGS in the MDCK cells that overexpress CaR[sup WT] inhibited extracellular Ca[sup 2+]-stimulated PLD activity, but pretreatment of cells with pertussis toxin and overexpression of RGS4 were without effect. The involvement of other signaling components such as protein kinase C, ADPribosylation factor, and phosphatidylinositol biphosphate was excluded. These findings demonstrate that the CaR couples to Gα[sub 12/13] to regulate PLD via a Rho-dependent mechanism and does so independently of Gα[sub i] and Gα[sub q]. This suggests that the CaR may regulate cytoskeleton via Gα[sub 12/13], Rho, and PLD. [ABSTRACT FROM AUTHOR]

In the Consortium on Resistance Against Carbapenems in Klebsiella and other Enterobacteriaceae (CRACKLE), trimethoprim-sulfamethoxazole (TMP-SMX) had a limited role in the treatment of less severe carbapenem-resistant Enterobacteriaceae (CRE) infections, especially urinary tract infections. Of tested CRE, only 29% were susceptible to TMP-SMX. Development of resistance further limits the use of TMP-SMX in CRE infections. [ABSTRACT FROM AUTHOR]

Hospital-acquired pneumonia represents one of the most difficult treatment challenges in infectious diseases. Many studies suggest that the timely administration of appropriate, pathogen-directed therapy can be lifesaving. Because results of culture and antimicrobial susceptibility testing can take 48 h or longer, physicians currently rely on clinical, epidemiological, and demographic factors to assist with the choice of empiric therapy for antibiotic-resistant pathogens. At present, a number of rapid molecular tests are being developed that identify pathogens and the presence of genetic determinants of antimicrobial resistance (eg, GeneXpert [Cepheid], ResPlex [Qiagen], FilmArray [Idaho Technologies], and Microarray [Check-Points]). In this review, the potential impact that molecular diagnostics has to identify and characterize pathogens that cause hospital-acquired bacterial pneumonia at an early stage is examined. In addition, a perspective on a novel technology, polymerase chain reaction followed by electrospray ionization mass spectrometry, is presented, and its prospective use in the diagnosis of pneumonia is also discussed. The complexities of the pulmonary microbiome represent a novel challenge to clinicians, but many questions still remain even as these technologies improve. [ABSTRACT FROM PUBLISHER]

Abstract: The predicted structure of the autosomal recessive polycystic kidney disease protein, fibrocystin, suggests that it may function as a receptor, but its function remains unknown. To understand its function, we searched for proteins that interact with the intracellular C-terminus of fibrocystin using the yeast two-hybrid system. From the screening, we found calcium modulating cyclophilin ligand (CAML), a protein involved in Ca2+ signaling. Immunofluorescent analysis showed that both proteins are co-localized in the apical membrane, primary cilia, and the basal body of cells derived from the distal nephron Epitope-tagged expression constructs of both proteins were co-immunoprecipitated from COS7 cells. The intracellular C-terminus of fibrocystin interacts with CAML, a protein with an intracellular distribution that is similar to that of PKD2. Fibrocystin may participate in regulation of intracellular Ca2+ in the distal nephron in a manner similar to PKD1 and PKD2 that are involved in autosomal dominant polycystic kidney disease. [Copyright &y& Elsevier]

In an effort to devise strategies for overcoming bacterial β-lactamases, we studied LN-1-255, a 6-alkylidene-2'-substituted penicillin sulfone inhibitor. By possessing a catecholic functionality that resembles a natural bacterial siderophore, LN-1-255 is unique among β-lactamase inhibitors. LN-1-255 combined with piperacillin was more potent against Escherichia coli DH10B strains bearing bla[subSHV] extended-spectrum and inhibitor-resistant β-lactamases than an equivalent amount of tazobactam and piperacillin. In addition, LN-1-255 significantly enhanced the activity of ceftazidime and cefpirome against extended-spectrum cephalosporin and Sme-1 containing carbapenem-resistant clinical strains. LN-1-255 inhibited SHV-1 and SHV-2 β-lactamases with nM affinity (K[sub1] = 110 ± 10 and 100 ± 10 nM, respectively). When LN-1-255 inactivated SHV β-lactamases, a single intermediate was detected by mass spectrometry. The crystal structure of LN-1-255 in complex with SHV-1 was determined at 1.55Å resolution. Interestingly, this novel inhibitor forms a bicyclic aromatic intermediate with its carbonyl oxygen pointing out of the oxyanion hole and forming hydrogen bonds with Lys-234 and Ser-130 in the active site. Electron density for the "tail" of LN-1-255 is less ordered and modeled in two conformations. Both conformations have the LN-1-255 carboxyl group interacting with Arg-244, yet the remaining tails of the two conformations diverge. The observed presence of the bicyclic aromatic intermediate with its carbonyl oxygen positioned outside of the oxyanion hole provides a rationale for the stability of this inhibitory intermediate. The 2'-substituted penicillin sulfone, LN-1-255, is proving to be an important lead compound for novel β-lactamase inhibitor design. [ABSTRACT FROM AUTHOR]

Bacterial resistance β-1actam/β-lactamase inhibitor combinations by single amino acid mutations in class A β-lactamases threatens our most potent clinical antibiotics. In TEM-1 and SHV-1, the common class A β-lactamases, alterations at Ser-130 confer resistance to inactivation by the β-lactamase inhibitors, clavulanic acid, and tazobactam. By using site-saturation mutagenesis, we sought to determine the amino acid substitutions at Ser-130 in SHV-1 β-lactamase that result in resistance to these inhibitors. Antibiotic susceptibility testing revealed that ampicillin and ampicillin/clavulanic acid resistance was observed only for the S130G β-lactamase expressed in Escherichia coli. Kinetic analysis of the S130G β-lactamase demonstrated a significant elevation in apparent K[sub m] and a reduction in k[sub cat]/K[sub m] for ampicillin. Marked increases in the dissociation constant for the preacylation complex, K[sub I], of clavulanic acid (SHV-1, 0.14 µM; S130G, 46.5 µM) and tazobactam (SHV-1, 0.07 µM; S130G, 4.2 µM) were observed. In contrast, the k[sub inact]s of S130G and SHV-1 differed by only 17% for clavulanic acid and 40% for tazobactam. Progressive inactivation studies showed that the inhibitor to enzyme ratios required to inactivate SHV-1 and S130G were similar. Our observations demonstrate that enzymatic activity is preserved despite amino acid substitutions that significantly alter the apparent affinity of the active site for β-lactams and β-lactamase inhibitors. These results underscore the mechanistic versatility of class A β-1actamases and have implications for the design of novel β-1actamase inhibitors. [ABSTRACT FROM AUTHOR]