Neisseria meningitidis is the cause of septicemia and meningococcal meningitis. During the course of infection, N. meningitidis encounters multiple environments within its host, which makes rapid adaptation to environmental changes a crucial factor for neisserial pathogenicity. Employing oligonucleotide-based DNA microarrays, we analyzed the transcriptome of N. meningitidis during two key steps of meningococcal infection, i.e., the interaction with epithelial cells (HeLa cells) and endothelial cells (human brain microvascular endothelial cells). Seventy-two genes were differentially regulated after contact with epithelial cells, and 48 genes were differentially regulated after contact with endothelial cells, including a considerable proportion of well-known virulence genes. While a considerable number of genes were in concordance between bacteria adherent to both cell types, we identified several open reading frames that were differentially regulated in only one system. The data obtained with this novel approach may provide insight into the pathogenicity mechanisms of N. meningitidis and could demonstrate the importance of gene regulation on the transcriptional level during different stages of meningococcal infection.

The current emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis calls for novel treatment strategies. Recently, BlaC, the principal ␤-lactamase of Mycobacterium tuberculosis, was recognized as a potential therapeutic target. The combination of meropenem and clavulanic acid, which inhibits BlaC, was found to be effective against even extensively drug-resistant M. tuberculosis strains when tested in vitro. Yet there is significant concern that drug resistance against this combination will also emerge. To investigate the potential of BlaC to evolve variants resistant to clavulanic acid, we introduced substitutions at important amino acid residues of M. tuberculosis BlaC (R220, A244, S130, and T237). Whereas the substitutions clearly led to in vitro clavulanic acid resistance in enzymatic assays but at the expense of catalytic activity, transformation of variant BlaCs into an M. tuberculosis H37Rv background revealed that impaired inhibition of BlaC did not affect inhibition of growth in the presence of ampicillin and clavulanate. From these data we propose that resistance to ␤-lactam-␤-lactamase inhibitor combinations will likely not arise from structural alteration of BlaC, therefore establishing confidence that this therapeutic modality can be part of a successful treatment regimen against M. tuberculosis.

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.

Mycobacterium abscessus is a highly drug-resistant nontuberculous mycobacterium (NTM). Efforts to discover new treatments for M. abscessus infections are accelerating, with a focus on cell wall synthesis proteins (M. abscessus l,d-transpeptidases 1 to 5 [LdtMab1 to LdtMab5] and d,d-carboxypeptidase) that are targeted by β-lactam antibiotics. A challenge to this approach is the presence of chromosomally encoded β-lactamase (BlaMab). Using a mechanism-based approach, we found that a novel ceftaroline-imipenem combination effectively lowered the MICs of M. abscessus isolates (MIC50 ≤ 0.25 μg/ml; MIC90 ≤ 0.5 μg/ml). Combining ceftaroline and imipenem with a β-lactamase inhibitor, i.e., relebactam or avibactam, demonstrated only a modest effect on susceptibility compared to each of the β-lactams alone. In steady-state kinetic assays, BlaMab exhibited a lower Ki app (0.30 ± 0.03 μM for avibactam and 136 ± 14 μM for relebactam) and a higher acylation rate for avibactam (k2/K = 3.4 × 105 ± 0.4 × 105 M−1 s−1 for avibactam and 6 × 102 ± 0.6 × 102 M−1 s−1 for relebactam). The kcat/Km was nearly 10-fold lower for ceftaroline fosamil (0.007 ± 0.001 μM−1 s−1) than for imipenem (0.056 ± 0.006 μM−1 s−1). Timed mass spectrometry captured complexes of avibactam and BlaMab, LdtMab1, LdtMab2, LdtMab4, and d,d-carboxypeptidase, whereas relebactam bound only BlaMab, LdtMab1, and LdtMab2. Interestingly, LdtMab1, LdtMab2, LdtMab4, LdtMab5, and d,d-carboxypeptidase bound only to imipenem when incubated with imipenem and ceftaroline fosamil. We next determined the binding constants of imipenem and ceftaroline fosamil for LdtMab1, LdtMab2, LdtMab4, and LdtMab5 and showed that imipenem bound >100-fold more avidly than ceftaroline fosamil to LdtMab1 and LdtMab2 (e.g., Ki app or Km of LdtMab1 = 0.01 ± 0.01 μM for imipenem versus 0.73 ± 0.08 μM for ceftaroline fosamil). Molecular modeling indicates that LdtMab2 readily accommodates imipenem, but the active site must widen to ≥8 Å for ceftaroline to enter. Our analysis demonstrates that ceftaroline and imipenem binding to multiple targets (l,d-transpeptidases and d,d-carboxypeptidase) and provides a mechanistic rationale for the effectiveness of this dual β-lactam combination in M. abscessus infections.

Background Toxic metabolic encephalopathy (TME) has been reported in 7–31% of hospitalized patients with coronavirus disease 2019 (COVID-19); however, some reports include sedation-related delirium and few data exist on the etiology of TME. We aimed to identify the prevalence, etiologies, and mortality rates associated with TME in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-positive patients. Methods We conducted a retrospective, multicenter, observational cohort study among patients with reverse transcriptase–polymerase chain reaction-confirmed SARS-CoV-2 infection hospitalized at four New York City hospitals in the same health network between March 1, 2020, and May 20, 2020. TME was diagnosed in patients with altered mental status off sedation or after an adequate sedation washout. Patients with structural brain disease, seizures, or primary neurological diagnoses were excluded. The coprimary outcomes were the prevalence of TME stratified by etiology and in-hospital mortality (excluding comfort care only patients) assessed by using a multivariable time-dependent Cox proportional hazards models with adjustment for age, race, sex, intubation, intensive care unit requirement, Sequential Organ Failure Assessment scores, hospital location, and date of admission. Results Among 4491 patients with COVID-19, 559 (12%) were diagnosed with TME, of whom 435 of 559 (78%) developed encephalopathy immediately prior to hospital admission. The most common etiologies were septic encephalopathy ( n = 247 of 559 [62%]), hypoxic-ischemic encephalopathy (HIE) ( n = 331 of 559 [59%]), and uremia ( n = 156 of 559 [28%]). Multiple etiologies were present in 435 (78%) patients. Compared with those without TME ( n = 3932), patients with TME were older (76 vs. 62 years), had dementia (27% vs. 3%) or psychiatric history (20% vs. 10%), were more often intubated (37% vs. 20%), had a longer hospital length of stay (7.9 vs. 6.0 days), and were less often discharged home (25% vs. 66% [all P < 0.001]). Excluding comfort care patients ( n = 267 of 4491 [6%]) and after adjustment for confounders, TME remained associated with increased risk of in-hospital death ( n = 128 of 425 [30%] patients with TME died, compared with n = 600 of 3799 [16%] patients without TME; adjusted hazard ratio [aHR] 1.24, 95% confidence interval [CI] 1.02–1.52, P = 0.031), and TME due to hypoxemia conferred the highest risk ( n = 97 of 233 [42%] patients with HIE died, compared with n = 631 of 3991 [16%] patients without HIE; aHR 1.56, 95% CI 1.21–2.00, P = 0.001). Conclusions TME occurred in one in eight hospitalized patients with COV...

Oligonucleotide-and cDNA-based microarrays comprising a subset of Neisseria meningitidis genes were assessed for study of the meningococcal heat shock response and found to be highly suitable for transcriptional profiling of N. meningitidis. Employing oligonucleotide arrays encompassing the entire genome of N. meningitidis, we analyzed the meningococcal heat shock response on a global scale and identified 55 heat shockderegulated open reading frames (34 induced and 21 repressed).Sequencing of the genomes of Neisseria meningitidis serogroup A and serogroup B strains provided us with a tremendously broad range of information (15,23). The next step is the elucidation of gene expression patterns and gene product function on a genome-wide scale. DNA microarrays offer an ideal tool for high-throughput investigation of gene regulation on the transcriptional level (for review, see references 4, 13, and 17). The two most commonly employed DNA microarray platforms are oligonucleotide and cDNA arrays. Here we performed a comparative analysis of the suitability of both technology platforms for transcriptional profiling of N. meningitidis.The two main features of DNA microarray performance are sensitivity (signal intensity) and specificity (ratio of specific to nonspecific hybridization). Additional care must be taken to standardize experimental conditions and to avoid the detection of false-positive signals (12). In order to validate gene expression modulations of N. meningitidis observed using cDNAbased and oligonucleotide-based microarrays, we first performed parallel hybridizations of identical RNA samples to the same slide. Microarrays containing probes specific for 60 genes selected from the published genome sequence of N. meningitidis serogroup B strain MC58 (23) were produced (Table 1). For cDNA-arrays, internal fragments of each open reading frame (ORF) (300 to 560 bp) were PCR amplified. For oligonucleotide arrays, oligonucleotides (40-mers, three per gene) comprising gene-specific internal fragments (covering 5Ј, central, and 3Ј parts) were designed. All oligonucleotides (manufactured by MWG-Biotech AG, Ebersberg, Germany) carried a C6 amino linker modification at the 5Ј end for covalent attachment to the slide surface. Each probe was spotted 5 (oligonucleotides) or 10 times (PCR products) per array using the Affymetrix 417 Arrayer (MWG-Biotech AG). PCR products were spotted on CMT-GAPS-Coated Slides (Corning, Wiesbaden, Germany), oligonucleotides were spotted on Super Aldehyde Slides (TeleChem International, Sunnyvale, Calif.), and the slides were processed according to the manufacturers' instructions.Cultures of N. meningitidis strain MC58 (24) were grown to mid-logarithmic growth phase (optical density at 600 nm [OD 600 ] ϭ 0.5/5 ϫ 10 8 CFU/ml) at 37°C in supplemented proteose peptone medium and RNA isolated as previously described (5). The RNA was split into two aliquots, and onehalf was labeled with Cy3-dCTP, the other with Cy5-dCTP (Amersham Pharmacia, Freiburg, Germany) during a firststrand reverse transcri...

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Durlobactam (DUR) is a potent inhibitor of Bla Mab and provides protection of amoxicillin and imipenem against hydrolysis. DUR has intrinsic activity and forms stable acyl-enzyme complexes with Ldt Mab2 and Ldt Mab4 . The ability of DUR to protect amoxicillin and imipenem against Bla Mab and its intrinsic activity along with the dual β-lactam target redundancy can explain the rationale behind the potent activity of this combination.