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Colistin resistance due to the mcr-type genes in Escherichia coli is well characterized. In order to study the resistance mechanism in mcr-negative colistin-resistant E. coli, strains were selected from a nationwide antimicrobial resistance surveillance program in Taiwan for further investigation. A total of 11 mcr-negative colistin-resistant isolates among 7,942 (0.1%) clinical E. coli isolates were identified between 2008 and 2018. Their prevalence was low and remained stable during the study period. Since 2012, ST131 and ST1193 clones with multiple drug-resistant phenotypes have emerged. All resistant strains displayed higher expression levels of the operons pmrHFIJKLM and pmrCAB than the control MG1655 strain. Although several amino acid substitutions were identified in PmrA or PmrB, only R81H in PmrA was associated with overexpression of pmrHFIJKLM and colistin resistance. The effect of substitution R81H in PmrA in colistin resistance was confirmed by complementation experiments. Although some strains harbored substitutions in PmrB, the identified mutations in pmrB did not contribute to colistin resistance. In conclusion, the amino acid substitution R81H in PmrA is an independent factor contributing to colistin resistance in non-mcr E. coli. IMPORTANCE The molecular epidemiology and resistance mechanisms of mcr-negative colistin-resistant E. coli are not well described. In this study, a total of 11 mcr-negative colistin-resistant E. coli isolates were selected from a nationwide antimicrobial resistance surveillance program in Taiwan for further investigation. We determined the resistance mechanism of non-mcr colistin-resistant strains using gene knockout and complementation experiments. We observed the occurrence of the global multiple-drug-resistant E. coli clones ST131 and ST1193 starting in 2012. Moreover, for the first time, we proved that the amino acid substitution R81H in PmrA is an independent factor contributing to colistin resistance in non-mcr E. coli. The study results helped to gain an insight into the diversity and complexity of chromosome-encoded colistin resistance in E. coli.

We report the first clinical Escherichia coli strain EC3000 with concomitant chromosomal colistin and carbapenem resistance. A novel in-frame deletion, Δ6-11 (RPISLR), in pmrB that contributes to colistin resistance was verified using recombinant DNA techniques. Although being less fit than the wild-type (WT) strain or EC3000 revertant (chromosomal replacement of WT pmrB in EC3000), a portion of serially passaged EC3000 strains preserving colistin resistance without selective pressure raises the concern for further spread.

We report the first clinical Escherichia coli strain EC3000 with concomitant chromosomal colistin and carbapenem resistance. A novel in-frame deletion, Δ6-11 (RPISLR), in pmrB that contributes to colistin resistance was verified using recombinant DNA techniques. Although being less fit than the wild-type (WT) strain or EC3000 revertant (chromosomal replacement of WT pmrB in EC3000), a portion of serially passaged EC3000 strains preserving colistin resistance without selective pressure raises the concern for further spread.

Cefoperazone, a third-generation cephamycin with broad-spectrum antibacterial activity and the ability to permeate bacterial cell membranes, is active against commonly encountered multidrug-resistant pathogens for hospital-acquired pneumonia (HAP) and health care-associated pneumonia (HCAP). To clarify the clinical effects of cefoperazone-sulbactam in the treatment of HAP and HCAP, we conducted an open-label, randomized, noninferiority trial that recruited patients aged ≥18 years suffering HAP/HCAP. Participants were randomly assigned to the cefoperazone-sulbactam (2 g of each per 12 h) or cefepime (2 g per 12 h) arm. Clinical and microbiological responses were evaluated at early posttherapy and test-of-cure visits. Recruited patients were allocated to subpopulations for intent-to-treat (n = 154), per-protocol (n = 147), and safety (n = 166) analyses. Intent-to-treat analysis demonstrated that (i) at the early posttherapy visit, 87.3% of patients receiving cefoperazone-sulbactam and 84.3% of patients receiving cefepime achieved clinical improvement or cure (risk difference of 3.0%; 95% confidence interval [CI], -9.0% to 15.0%), and (ii) at the test-of-cure visit, 73.1% of patients receiving cefoperazone-sulbactam and 56.8% of patients receiving cefepime were assessed as cured (risk difference of 16.3%; 95% CI, 0.0% to 33.0%). These results indicated the noninferiority of cefoperazone-sulbactam to cefepime, which was confirmed by per-protocol analysis. The chest radiographic consolidation/infiltration resolution rate, microbiological eradiation rate, and percentage of adverse events were comparable in both groups. Serious adverse events were rare, and none was judged to be related to the study drugs. Cefoperazone-sulbactam at 2 g every 12 h was noninferior to cefepime at 2 g every 2 h for patients with HCAP.

Acinetobacter baumannii biofilms are difficult to eradicate. We investigated the effects of meropenem (2 mg/liter), imipenem (2 mg/liter), sulbactam (4 mg/liter), colistin (2 mg/liter), and tigecycline (2 mg/liter), alone or in combination, on biofilm-embedded carbapenem-resistant and carbapenem-susceptible A. baumannii (CRAb and CSAb, respectively) cells, as well as on the architecture of the biofilms. A. baumannii ATCC 15151 (Ab15151) and its OXA-82-overproducing transformant, along with two clinical CSAb and two clinical CRAb isolates of differing clonalities, were used. The minimal bactericidal concentrations for biofilm-embedded cells of the six tested isolates were >50-fold those of their planktonic cells. When used individually, meropenem exhibited a higher killing effect than the other four antimicrobials on biofilm-embedded CSAb cells in the colony biofilm assay. For two clinical CRAb isolates, meropenem plus sulbactam or sulbactam plus tigecycline showed >100-fold the bactericidal effect exhibited by these agents used alone after 48 h of treatment. The effect of antimicrobials on the architecture of Ab15151 biofilm emitting green fluorescence was determined by confocal laser scanning microscopy using COMSTAT software. Significant decreases in the maximum biofilm thickness were observed after exposure to meropenem and imipenem. Meropenem plus sulbactam significantly decreased the biomass and mean thickness and increased the roughness coefficient of biofilms, but sulbactam plus tigecycline only decreased the maximum and mean biofilm thickness compared to any of these agents used alone. Meropenem was active against biofilm-embedded CSAb, whereas meropenem plus sulbactam exhibited synergism against biofilm-embedded CRAb and caused significantly more damage to the biofilm architecture than did any of the agents used alone.

In this study, a novel colloidal gold-based immunochromatographic strip (ICS) containing anti- Klebsiella pneumoniae capsular polysaccharide polyclonal antibodies was developed to specifically detect K. pneumoniae serotypes K1 and K2. Capsular polysaccharide K1 and K2 antigens were first used to produce polyclonal anti-K1 and anti-K2 antibodies. Reference strains with different serotypes, nontypeable K. pneumoniae strains, and other bacterial species were then used to assess the sensitivity and specificity of these test strips. The detection limit was found to be 10 5 CFU, and the ICSs were stable for 6 months when stored at room temperature. No false-positive or false-negative results were observed, and equivalent results were obtained compared to those of more conventional test methods, such as PCR or serum agglutination. In conclusion, the ICS developed here requires no technical expertise and allows for the specific, rapid, and simultaneous detection of K. pneumoniae serotypes K1 and K2.

Capsular serotypes, magA , and rmpA have been documented in high prevalence for Klebsiella pneumoniae liver abscess. To investigate the regional difference and the correlation of capsular serotype, magA , and rmpA with virulence, 73 isolates were collected in Singapore and Taiwan. Capsular serotypes were determined by countercurrent immunoelectrophoresis, the presence of magA and rmpA was determined by PCR, and virulence was determined by phagocytosis and mouse inoculation. Isolates from Singapore were similar to those from Taiwan in genomic heterogeneity, prevalence of serotype, and the presence of magA and rmpA. The most common serotype was K1 (34/73; 46.6%), followed by K2 (15/73; 20.5%). magA was restricted to serotype K1. All K1 or K2 isolates and 66.7% (16/24) of isolates that were neither serotype K1 nor serotype K2 (non-K1/K2) carried rmpA . Serotype K1 or K2 isolates demonstrated significantly more phagocytic resistance and virulence than did rmpA -positive and -negative groups of non-K1/K2 isolates. In the non-K1/K2 group, the virulence profiles of rmpA -positive strains from Taiwan and Singapore were different by phagocytosis assay and in the mouse model, indicating that factors other than rmpA contributed to virulence. The characteristics of K. pneumoniae liver abscess in Singapore and Taiwan are similar. Capsular serotype K1 or K2 plays a more important role than magA and rmpA in determining virulence in K. pneumoniae liver abscess.

Reported prevalences of human herpesvirus 8 (HHV-8) (Kaposi’s sarcoma-associated herpesvirus) in semen have ranged widely. This is possibly due to differences in assay sensitivity, geographic or population-based differences in the true presence of the virus in semen, and PCR contamination. This study assessed interlaboratory sensitivity and reproducibility in the analysis of blinded experimental panels, each consisting of 48 specimens and being composed of semen specimens from different healthy artificial-insemination donors ( n = 30) and human immunodeficiency virus (HIV)-infected patients ( n = 7) plus positive ( n = 4) and negative ( n = 7) controls. The experimental panels analyzed in each laboratory were identical except for being independently coded. Of 10 experiments done in five laboratories, 5 experiments from three laboratories had evidence of PCR contamination; all instances of contamination were in the context of nested PCR procedures. In the experiments with no false-positive results, HHV-8 DNA was detected in three (8%) of the 37 semen specimens (two from artificial-insemination donors and one from an HIV-positive patient) but in only 3 (1.6%) of the 184 PCRs in which these specimens were analyzed. This suggests that HHV-8 DNA is present in semen at concentrations that can be too low to allow its consistent detection. This study emphasizes the importance of performing blinded, multi-institution experiments to provide a coherent basis for comparing results and to motivate standardization of methods.

(S)-9-(3-Hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA], (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)-2,6-diaminopurine [(S)-HPMPDAP], cyclic (S)-HPMPA [(S)-cHPMPA], 9-(2-phosphonylmethoxyethoxyethyl)-2,6-diaminopurine (PMEDAP), and (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine [(S)-HPMPC] were examined for their inhibitory effects on Epstein-Barr virus (EBV) replication. The 50% effective concentrations for inhibition of viral DNA replication were 0.16, 0.03, 2.0, 1.5, and 0.08 microM for PMEDAP, (S)-HPMPC, (S)-HPMPDAP, (S)-cHPMPA, and (S)-HPMPA, respectively. The relative efficacies based on the in vitro therapeutic index was (S)-HPMPC (5,000) greater than PMEDAP (1,000) = (S)-HPMPA (1,000) greater than (S)-cHPMPA (136) greater than (S)-HPMPDAP (78). Certain ratios of combinations of (S)-HPMPC with 3'-azido-3'-deoxythymidine produced a synergistic inhibitory effect on EBV genome replication, but others exhibited an antagonistic effect. These results indicate that this series of acyclic nucleoside phosphonate analogs, and in particular (S)-HPMPC, are potent and selective anti-EBV agents in vitro.

In order to identify the gene encoding the Epstein-Barr virus (EBV) DNA polymerase, a portion of the BamHI-A fragment containing the fifth leftward open reading frame (BALF5) of the EBV genome was cloned into SP6 and T7 promoter-containing vectors for in vitro transcription-translation. The RNA synthesized in vitro was used to program rabbit reticulocyte lysates, which were analyzed for the synthesis of the putative polymerase polypeptide (110 kDa) and assayed directly for EBV DNA polymerase activity. The polypeptide synthesized by the full-length BALF5 genomic fragment had a molecular mass of 110 kDa. 5'-truncated BALF5 with the first and second ATGs deleted produced 95- and 83-kDa polypeptides, respectively. All three translation products were enzymatically active and displayed resistance to high salt concentrations. The identity of the largest polypeptide as the viral polymerase was established by (i) immunoprecipitation with EBV-positive sera from patients with nasopharyngeal carcinoma and by a rabbit polyclonal antiserum prepared with a synthetic peptide derived from the DNA sequence of BALF5; (ii) identification of a polypeptide of identical size (110 kDa) immunoprecipitated from superinfected Raji cell extracts by these antibodies; and (iii) salt-resistant enzymatic activity which was neutralized by the rabbit EBV antiserum. Thus, BALF5 encodes a functional polymerase identical to that induced in superinfected Raji cells.

Klebsiella pneumoniae has emerged as the leading cause of community-acquired pyogenic liver abscess in many countries. Community-acquired pyogenic liver abscess due to K. pneumoniae with a unique antibiogram indicative of resistance only to ampicillin has been demonstrated. We reported the first

We report a case of acute cholecystitis accompanied by acute pancreatitis and caused by Dolosigranulum pigrum in a 76-year-old male with gallstones. D. pigrum was isolated from a blood culture and confirmed by biochemistry tests and 16S rRNA sequencing. The isolate was susceptible to the β-lactams ampicillin, penicillin, cephalothin, ceftriaxone, ceftazidime, chloramphenicol, and vancomycin but was intermediate to erythromycin and clindamycin. The patient recovered without sequelae after treatment with appropriate antibiotics for two weeks.

We report the use of monoclonal antibody against the early antigen diffuse component (anti-EA-D) of Epstein-Barr virus (EBV) to analyze, both qualitatively and quantitatively, the expression of EA-D in various human lymphoblastoid cell lines activated by chemical inducers. The kinetics of synthesis of EA-D in P3HR-1, B95-8, and Ramos/AW cells were similar in that they all reached the peak of synthesis on day 5 after induction. Surprisingly, no expression of EA-D was found in induced BJAB/GC, an EBV-genome-containing cell line. EBV-negative cell lines, BJAB and Ramos, were negative for EA-D. Raji cells had no detectable EA-D but responded rapidly to induction, reaching a peak on day 3. Superinfection of Raji cells also resulted in marked induction of EA-D, which reached a plateau between 8 to 12 h postinfection. Western blotting coupled with the enzyme-linked immunosorbent assay was employed to identify polypeptides representing EA-D. A family of four polypeptides with molecular weights of 46,000 (46K protein), 49,000, 52,000, and 55,000 were identified to be reactive with monoclonal anti-EA-D antiserum. The pattern of EA-D polypeptides expressed in each cell line was different. Of particular interest was the expression of a large quantity of 46K protein both in induced Raji and P3HR-1 cells, but not in superinfected Raji cells. A 49K doublet was expressed in activated p3HR-1, B95-8, and Ramos/AW cells and in superinfected Raji cells. In addition, two distinct 52K and 55K polypeptides were expressed in induced Ramos/AW and superinfected Raji cells. However, none of these EA-D polypeptides was detectable in BJAB/GC, BJAB, Ramos, and mock-infected Raji cells. To approximate relative concentrations of EA-D in cell extracts, we employed the enzyme-linked immunosorbent assay and immunoblot dot methods by using one of the purified EA-D components to construct a standard curve. Depending upon the cell lines, it was estimated that ca. 1 to 3% (determined by the enzyme-linked immunosorbent assay) and 0.8 to 1.6% (determined by immunoblot dot) of total proteins from maximally induced cells were EA-D. These results suggest that differential expression of EA-D polypeptides could be of importance in the diagnosis of state of EBV infection.

We investigated the effects of acyclovir and 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) on a lymphoblastoid cell line dually infected with Epstein-Barr virus and herpes simplex virus (HSV) type 1. The numbers of Epstein-Barr virus genomes were reduced during 70 days of treatment with either drug. Both drugs suppressed HSV replication in a dose-related manner. In the continued presence of the drugs, HSV developed resistance, rapidly to acyclovir and much more slowly to 30 microM DHPG. Analysis of HSV glycoprotein C production and viral DNA showed that treatment with 100 microM DHPG eliminated HSV production, curing the cell line of HSV persistent infection.

The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) is a potent inducer of Epstein-Barr virus (EBV) gene expression. The optimal conditions for maximum activation of latent EBV genomes by TPA were determined. Although TPA is able to induce replication of EBV genomes in P3HR-1 cells in all phases of growth, the greatest increase in viral genome copies per cell (15-fold above the control level) occurred in nonproliferating cells as opposed to cells growing exponentially (6-fold above the control level). The synthesis of chromosomal proteins in nonproliferating cells under the conditions that induce maximum activation of latent virus genomes by TPA was studied. Selective stimulation in chromosomal protein synthesis accompanied the increase in EBV genomes in P3HR-1 cells despite an overall reduction in total cellular protein synthesis. Comparison of the chromosomal proteins from TPA-induced P3HR-1 cells and from superinfected Raji cells revealed comigrating chromosomal polypeptides of 145K, 140K, 135K, 110K, 85K, and 55K that are presumably EBV associated. The selective stimulation of synthesis of these chromosomal proteins in TPA-treated P3HR-1 cells was closely associated with the activation of latent EBV genomes.

The heterogeneity of Epstein-Barr virus (EBV) obtained from P3HR-1 cells has permitted derivation of a distinct subclone of P3HR-1 (L. Heston, M. Rabson, N. Brown, and G. Miller, Nature (London) 295:160-163, 1982). We have analyzed the biologic properties and genomic structure of this subclonal virus (clone 13) compared with those of parental P3HR-1 and B95-8 viruses. Synthesis of EBV compared with those of parental P3HR-1 and B95-8 viruses. Synthesis of EBV proteins in Raji cells superinfected with virus derived from P3HR-1, clone 13, and B95-8 was analyzed both by fluorography of radiolabeled proteins and by immunoblotting. Highly concentrated preparations of clone 13 and B95-8 virus induced most of the spectrum of EBV proteins in Raji cells with the exception of the 145,000-, 140,000-, and 110,000-molecular-weight proteins, which were either undetectable or reduced. Moreover, both clone 13 and B95-8 viruses also induced the same patterns of early antigen diffuse components as the parental P3HR-1 virus did. However, only P3HR-1 virus could induce EBV DNA synthesis in superinfected Raji cells, as determined both by buoyant density centrifugation and by in situ cytohybridization with biotinylated recombinant EBV DNA probes. Defective heterogeneous molecules present in P3HR-1 virus have been implicated in early antigen induction after superinfection of Raji cells. Therefore, Southern blots of clone 13, P3HR-1, and B95-8 viruses were hybridized to recombinant EBV fragments representing the sequences contained within the defective molecules in P3HR-1. The parental P3HR-1 contained the previously described defective molecules. No evidence for defective molecules was found in clone 13 or B95-8 viruses. These data indicate that concentrated preparations of both clone 13 and B95-8 viruses can induce abortive infection in Raji cells, but while the defective molecules are not needed for induction of early antigen diffuse components, they may be required for the induction of viral DNA synthesis.

The Epstein-Barr virus (EBV) BZLF1 gene product is thought to mediate the disruption of latent EBV infection. We have examined the regulatory effects of BZLF1 by studying its transactivating effects on seven different EBV promoters. We find that whereas the BZLF1 gene product increases the activity of the two early promoters, BMLF1 and BMRF1, it decreases the activity of three latent promoters (the BamHI-C and BamHI-W Epstein-Barr nuclear antigen promoters and the latent membrane protein promoter). The BZLF1-induced changes in promoter-directed chloramphenicol acetyltransferase activity occur in EBV-negative as well as EBV-positive cell lines and are accompanied by a similar change in chloramphenicol acetyltransferase mRNA. Deletion analysis of the BamHI Z fragment indicates that in a portion of the amino-terminal half of the BZLF1 gene product (amino acids 24 to 86) is not essential for positive transactivating effects but is required for down-regulating effects. Thus, different domains of the same EBV immediate-early gene product can either increase the function of EBV promoters active in productive infection or decrease the function of key promoters active in latent infection.

The effect of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU), a new antiviral drug, on Epstein-Barr virus (EBV) was studied and compared with those of E-5-(2-bromovinyl)-2'-deoxyuridine (BVdU) and acyclovir (ACV). BV-araU effectively inhibited EBV replication both in superinfected Raji cells and in virus producer P3HR-1(LS) cells, as determined by density gradient centrifugation, in situ cytohybridization with an EBV DNA probe, and cRNA-DNA hybridization. The 50% effective doses for viral DNA replication were 0.26, 0.06, and 0.3 microM for BV-araU, BVdU, and ACV, respectively. The relative efficacy on the basis of the in vitro therapeutic index was BVdU (6,500) greater than BV-araU (1,500) greater than ACV (850). Synthesis of EBV-induced polypeptides with molecular weights of 145,000 and 140,000 was inhibited by these drugs. Kinetic analysis of reversibility of inhibition of EBV DNA replication after removal of the drugs indicated that BV-araU, like BVdU, has a more prolonged inhibitory effect than ACV. These results indicate that the 2' OH group in the arabinosyl configuration of BV-araU results in marked reduction in anti-EBV activity while slightly diminishing cytotoxicity.

We show that the anti-human immunodeficiency virus agent, 3'-azido-3'-deoxythymidine (AZT), which suppresses infectivity and cytopathic effects of human immunodeficiency virus, also effectively inhibits Epstein-Barr virus (EBV) DNA replication. However, AZT has no effect on four other human herpesviruses: cytomegalovirus, varicella-zoster virus, and herpes simplex virus types 1 and 2. The combination of acyclovir and AZT, while it is not synergistic, has an additive effect against EBV replication. AZT may prove to be a useful drug for treatment of coinfections with human immunodeficiency virus and EBV.

A dual antibody probing technique that permitted a color-coded identification of polypeptides representing different classes of Epstein-Barr virus (EBV) antigens as well as differentiation of the polypeptides induced by different herpesviruses in the same Western blot was developed. When the nitrocellulose sheet was probed first with monoclonal antibody against EBV early antigen diffuse component (EA-D) and then stained with 4-chloro-1-naphthol, four polypeptides specific for EA-D were identified by purple bands. Subsequently, the same nitrocellulose sheet was reprobed with human serum containing antibodies against EBV early antigen, viral capsid antigen, and nuclear antigen and stained with 3,3'-diaminobenzidine. Several brown bands corresponding to early, viral capsid, and nuclear antigen polypeptides were detected. The dual antibody probing technique was used in an analysis to differentiate polypeptides resulting from either EBV or herpes simplex virus infection, either in cells infected by individual virus or in a cell line dually infected by both viruses. On the basis of different colored bands in different lanes of the same gel, 20 polypeptides with molecular weights ranging from 31,000 to 165,000 were identified as herpes simplex virus-specific proteins. These results suggested that the dual antibody probing technique may be applicable in clinical diagnosis for detecting antigens and antibodies derived from different pathogens.

The effects of (2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC), 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-methyluridine (FMAU), 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouridine (FIAU), (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdU), and 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG or BW B759U) on the replication of Epstein-Barr virus (EBV) in vitro were evaluated and compared with that of acyclovir (ACV). The relative potencies of these drugs, on the basis of anti-EBV activity, were: FIAC = FIAU greater than FMAU greater than DHPG greater than BVdU greater than ACV; on the basis of the therapeutic index they were: BVdU greater than DHPG greater than FIAC greater than ACV greater than FIAU greater than FMAU. Differential inhibition of EBV-associated polypeptides by these drugs was observed.