Crystal structures of apo OXA-58 with a static Ω-loop and the flexible loops of the substrate-binding cleft.

<p>Left, the surface representation of the substrate-binding cleft in closed state (A, green), and open state (B, orange) in a previously reported OXA-58 crystal structure (PDB ID: 4OH0). Right, superimposed models of the two crystal structures of OXA-58; closed state (green) and open state (orange). The dashed lines represent hydrogen bonds for the ζ-carbamic acid of Lys86 connect to the Ω-loop from the loop containing Ser83. The white model represents a acyl-intermediate meropenem structure superimposed with OXA-23 (PDB ID: 4JF4) as a prospected carbapenem binding.</p

The closed substrate-binding cleft of OXA-58.

<p>A, A representation of the overall OXA-58 structure. The loops forming the substrate-binding cleft are colored green. B, A representation of the overall OXA-58 surface shown in the same colors as in panel A. C, The hydrogen bond network in the active site. Dashed line connections in stick models indicate hydrogen bonds between each atom from the ζ carbamic acid of Lys86 and Ser83. The purple meshes are a <i>F</i>_o−<i>F</i>_c omit map of the residues involved in the hydrogen bond network that includes bound water molecules with contouring at the 3<i>σ</i> level. The arch-like roof architecture is represented using a transparent molecular surface envelope, and the residues forming the architecture are shown in the stick model.</p

Stabilization of OXA-58 concomitant with activation in NaHCO₃.

<p>The bar graphs in the left column show the residual activities of wild-type (wt, A), W169A (B), and S83A (C) after heat treatment. The white bars represent the specific activities of enzymes stored at 4°C without heat treatment in 100 mM NaHCO₃ as a control. The black bars are the residual activities after heat treatment in presence of NaHCO₃ at the concentrations indicated. The graphs of plots and curves in the left column are the specific activities of the wild-type (D), W169A (E), and S83A (F) enzymes vs. NaHCO₃ concentrations on a logarithmic scale.</p

Epidemiology of Extended-Spectrum β-Lactamase Producing <i>Escherichia coli</i> in the Stools of Returning Japanese Travelers, and the Risk Factors for Colonization

<div><p>Objective</p><p>Travel overseas has recently been considered a risk factor for colonization with drug-resistant bacteria. The purpose of this study was to establish the epidemiology and risk factors associated with the acquisition of drug-resistant bacteria by Japanese travelers.</p><p>Methods</p><p>Between October 2011 and September 2012, we screened the stools of 68 Japanese returning travelers for extended-spectrum β-lactamase (ESBL) producing <i>Escherichia coli</i>. All specimens were sampled for clinical reasons. Based on the results, the participants were divided into an ESBL-producing <i>E. coli</i> positive group (18 cases; 26%) and an ESBL-producing <i>E. coli</i> negative group (50 cases; 74%), and a case-control study was performed. Microbiological analyses of ESBL-producing strains, including susceptibility tests, screening tests for metallo-β-lactamase, polymerase chain reaction amplification and sequencing of <i>bla</i>_CTX-M genes, multilocus sequence typing, and whole genome sequencing, were also conducted.</p><p>Results</p><p>In a univariate comparison, travel to India was a risk factor (Odds Ratio 13.6, 95% Confidence Interval 3.0–75.0, p<0.0001). There were no statistical differences in the characteristics of the travel, such as backpacking, purpose of travel, interval between travel return and sampling stool, and duration of travel. Although 10 of 13 analyzed strains (77%) produced CTX-M-15, no ST131 clone was detected.</p><p>Conclusion</p><p>We must be aware of the possibilities of acquiring ESBL-producing <i>E. coli</i> during travel in order to prevent the spread of these bacteria not only in Japan but globally.</p></div

The characteristics of ESBL-producing <i>E. coli</i> positive/negative groups.

1)<p>Student’s t-test, ²⁾ Fisher’s exact test.</p><p>ESBL = extended-spectrum β-lactamase; SD = standard deviation; VFR = visiting friends and relatives;</p

The characteristics of ESBL-producing <i>E. coli</i> positive/negative groups in travelers to India.

1)<p>Student’s t-test, ²⁾ Fisher’s exact test.</p><p>ESBL = extended-spectrum β-lactamase; SD = standard deviation; VFR = visiting friends and relatives;</p

Clinical Specimen-Direct LAMP: A Useful Tool for the Surveillance of <i>bla</i>_OXA-23-Positive Carbapenem-Resistant <i>Acinetobacter baumannii</i>

<div><p>Healthcare-associated infections are a leading cause of morbidity and mortality worldwide. Treatment is increasingly complicated by the escalating incidence of antimicrobial resistance. Among drug-resistant pathogens, carbapenem-resistant <i>Acinetobacter baumannii</i> (CRAb) is of increasing concern because of the limited applicable therapies and its expanding global distribution in developed countries and newly industrialized countries. Therefore, a rapid detection method that can be used even in resource-poor countries is urgently required to control this global public health threat. Conventional techniques, such as bacterial culture and polymerase chain reaction (PCR), are insufficient to combat this threat because they are time-consuming and laborious. In this study, we developed a loop-mediated isothermal amplification (LAMP) method for detecting <i>bla</i>_OXA-23-positive CRAb, the most prevalent form of CRAb in Asia, especially in Thailand, and confirmed its efficacy as a surveillance tool in a clinical setting. Clinical samples of sputum and rectal swabs were collected from patients in a hospital in Bangkok and used for LAMP assays. After boiling and centrifugation, the supernatants were used directly in the assay. In parallel, a culture method was used for comparison purposes to evaluate the specificity and sensitivity of LAMP. As a first step, a total of 120 sputum samples were collected. The sensitivity of LAMP was 88.6% (39/44), and its specificity was 92.1% (70/76) using the culture method as the “gold standard”. When surveillance samples including sputum and rectal swabs were analyzed with the LAMP assay, its sensitivity was 100.0%. This method enables the direct analysis of clinical specimens and provides results within 40 minutes of sample collection, making it a useful tool for surveillance even in resource-poor countries.</p></div

Genotypes of carbapenem-resistant <i>A</i>. <i>baumannii</i> (CRAb) isolates from hospital patients in Bangkok.

<p>Total^a: Total number of isolates with each resistance gene</p><p>%^b: The proportion of isolates with each resistance gene</p><p>Genotypes of carbapenem-resistant <i>A</i>. <i>baumannii</i> (CRAb) isolates from hospital patients in Bangkok.</p

LAMP primers used for <i>bla</i>_oxa-23 and the ITS sequence.

<p>F3: outer forward primer; B3: backward inner primer; LF/LB: loop primersouter backward primer; FIP: forward inner primer; BIP:</p><p>LAMP primers used for <i>bla</i>_oxa-23 and the ITS sequence.</p

Real-time turbidity assays under various conditions using a turbidimeter.

<p>(A) To determine the optimal reaction conditions, a LAMP assay was performed on extracted bacterial DNA at temperatures ranging from 62°C to 67°C. At 65°C, the reaction finished within the shortest period of time, and the negative control remained transparent after 60 minutes of incubation. (B) To determine the detection limit, the extracted DNA templates were serially diluted 10 times (from 2 pg to 2×10⁻⁶ pg) and used in the LAMP assay. The turbidity was evaluated with a turbidimeter every 5 minutes.</p