Determination of the target nucleosides for members of two families of 16S rRNA methyltransferases that confer resistance to partially overlapping groups of aminoglycoside antibiotics

The 16S ribosomal RNA methyltransferase enzymes that modify nucleosides in the drug binding site to provide self-resistance in aminoglycoside-producing micro-organisms have been proposed to comprise two distinct groups of S-adenosyl-l-methionine (SAM)-dependent RNA enzymes, namely the Kgm and Kam families. Here, the nucleoside methylation sites for three Kgm family methyltransferases, Sgm from Micromonospora zionensis, GrmA from Micromonospora echinospora and Krm from Frankia sp. Ccl3, were experimentally determined as G1405 by MALDI-ToF mass spectrometry. These results significantly extend the list of securely characterized G1405 modifying enzymes and experimentally validate their grouping into a single enzyme family. Heterologous expression of the KamB methyltransferase from Streptoalloteichus tenebrarius experimentally confirmed the requirement for an additional 60 amino acids on the deduced KamB N-terminus to produce an active methyltransferase acting at A1408, as previously suggested by an in silico analysis. Finally, the modifications at G1405 and A1408, were shown to confer partially overlapping but distinct resistance profiles in Escherichia coli. Collectively, these data provide a more secure and systematic basis for classification of new aminoglycoside resistance methyltransferases from producers and pathogenic bacteria on the basis of their sequences and resistance profiles

Prevalence, risk factors, and impact on outcome of cytomegalovirus replication in serum of Cambodian HIV-infected patients (2004-2007)

BACKGROUND: In developing countries, the study of cytomegalovirus (CMV) coinfection in HIV-infected patients remains neglected. Quantitative CMV polymerase chain reaction (PCR) is the gold standard diagnostic tool for analyzing serum CMV replication and for predicting CMV disease. We estimated the prevalence of replicating CMV in sera of newly diagnosed HIV-infected Cambodian patients and examined its impact on mortality. METHODS: This cohort study was based on 2 highly active antiretroviral therapy treatment programs in Cambodia between 2004 and 2007. Quantitative CMV PCR was performed on baseline serum samples of 377 HIV-infected patients. RESULTS: The prevalence of serum CMV DNA was 55.2% (150 of 272) in patients with CD4 count <100/mm. In multivariate analysis, hemoglobin <9 g/dL, CD4 count <100/mm, and Karnofsky index <50 were independently associated with positive serum CMV DNA at baseline. During a 3-year follow-up period, CMV viral load >or=3.1 log10 copies per milliliter was significantly associated with death independently of CD4 count, other opportunistic infections, and highly active antiretroviral therapy. CONCLUSIONS: As in industrialized countries, serum CMV replication is highly prevalent among HIV-infected Cambodian patients and is associated with increased mortality. This underscores the importance of diagnostic CMV infection by PCR in sera of HIV-infected patients with CD4 count <100/mm and treating this opportunistic infection to reduce its associated mortality

Lovastatin Protects against Experimental Plague in Mice

Background: Plague is an ectoparasite-borne deadly infection caused by Yersinia pestis, a bacterium classified among the group A bioterrorism agents. Thousands of deaths are reported every year in some African countries. Tetracyclines and cotrimoxazole are used in the secondary prophylaxis of plague in the case of potential exposure to Y. pestis, but cotrimoxazole-resistant isolates have been reported. There is a need for additional prophylactic measures. We aimed to study the effectiveness of lovastatin, a cholesterol-lowering drug known to alleviate the symptoms of sepsis, for plague prophylaxis in an experimental model. Methodology: Lovastatin dissolved in Endolipide was intraperitoneally administered to mice (20 mg/kg) every day for 6 days prior to a Y. pestis Orientalis biotype challenge. Non-challenged, lovastatin-treated and challenged, untreated mice were also used as control groups in the study. Body weight, physical behavior and death were recorded both prior to infection and for 10 days post-infection. Samples of the blood, lungs and spleen were collected from dead mice for direct microbiological examination, histopathology and culture. The potential antibiotic effect of lovastatin was tested on blood agar plates. Conclusions/Significance: Lovastatin had no in-vitro antibiotic effect against Y. pestis. The difference in the mortality between control mice (11/15; 73.5%) and lovastatin-treated mice (3/15; 20%) was significant (P,0.004; Mantel-Haensze

High prevalence of plasmid-mediated 16S rRNA methylase gene rmtB among Escherichia coli clinical isolates from a Chinese teaching hospital

<p>Abstract</p> <p>Background</p> <p>Recently, production of 16S rRNA methylases by Gram-negative bacilli has emerged as a novel mechanism for high-level resistance to aminoglycosides by these organisms in a variety of geographic locations. Therefore, the spread of high-level aminoglycoside resistance determinants has become a great concern.</p> <p>Methods</p> <p>Between January 2006 and July 2008, 680 distinct <it>Escherichia coli </it>clinical isolates were collected from a teaching hospital in Wenzhou, China. PCR and DNA sequencing were used to identify 16S rRNA methylase and extended-spectrum β-lactamase (ESBL) genes, including <it>armA </it>and <it>rmtB</it>, and in situ hybridization was performed to determine the location of 16S rRNA methylase genes. Conjugation experiments were subsequently performed to determine whether aminoglycoside resistance was transferable from the <it>E. coli </it>isolates via 16S rRNA methylase-bearing plasmids. Homology of the isolates harboring 16S rRNA methylase genes was determined using pulse-field gel electrophoresis (PFGE).</p> <p>Results</p> <p>Among the 680 <it>E. coli </it>isolates, 357 (52.5%), 346 (50.9%) and 44 (6.5%) isolates were resistant to gentamicin, tobramycin and amikacin, respectively. Thirty-seven of 44 amikacin-resistant isolates harbored 16S rRNA methylase genes, with 36 of 37 harboring the <it>rmtB </it>gene and only one harboring <it>armA</it>. The positive rates of 16S rRNA methylase genes among all isolates and amikacin-resistant isolates were 5.4% (37/680) and 84.1% (37/44), respectively. Thirty-one isolates harboring 16S rRNA methylase genes also produced ESBLs. In addition, high-level aminoglycoside resistance could be transferred by conjugation from four <it>rmtB</it>-positive donors. The plasmids of incompatibility groups IncF, IncK and IncN were detected in 34, 3 and 3 isolates, respectively. Upstream regions of the <it>armA </it>gene contained <it>IS</it>CR1 and <it>tnpU</it>, the latter a putative transposase gene,. Another putative transposase gene, <it>tnpD</it>, was located within a region downstream of <it>armA</it>. Moreover, a transposon, Tn<it>3</it>, was located upstream of the <it>rmtB</it>. Nineteen clonal patterns were obtained by PFGE, with type H representing the prevailing pattern.</p> <p>Conclusion</p> <p>A high prevalence of plasmid-mediated <it>rmtB </it>gene was found among clinical <it>E. coli </it>isolates from a Chinese teaching hospital. Both horizontal gene transfer and clonal spread were responsible for the dissemination of the <it>rmtB </it>gene.</p

Novel Genetic Tools for Diaminopimelic Acid Selection in Virulence Studies of Yersinia pestis

Molecular studies of bacterial virulence are enhanced by expression of recombinant DNA during infection to allow complementation of mutants and expression of reporter proteins in vivo. For highly pathogenic bacteria, such as Yersinia pestis, these studies are currently limited because deliberate introduction of antibiotic resistance is restricted to those few which are not human treatment options. In this work, we report the development of alternatives to antibiotics as tools for host-pathogen research during Yersinia pestis infections focusing on the diaminopimelic acid (DAP) pathway, a requirement for cell wall synthesis in eubacteria. We generated a mutation in the dapA-nlpB(dapX) operon of Yersinia pestis KIM D27 and CO92 which eliminated the expression of both genes. The resulting strains were auxotrophic for diaminopimelic acid and this phenotype was complemented in trans by expressing dapA in single and multi-copy. In vivo, we found that plasmids derived from the p15a replicon were cured without selection, while selection for DAP enhanced stability without detectable loss of any of the three resident virulence plasmids. The dapAX mutation rendered Y. pestis avirulent in mouse models of bubonic and septicemic plague which could be complemented when dapAX was inserted in single or multi-copy, restoring development of disease that was indistinguishable from the wild type parent strain. We further identified a high level, constitutive promoter in Y. pestis that could be used to drive expression of fluorescent reporters in dapAX strains that had minimal impact to virulence in mouse models while enabling sensitive detection of bacteria during infection. Thus, diaminopimelic acid selection for single or multi-copy genetic systems in Yersinia pestis offers an improved alternative to antibiotics for in vivo studies that causes minimal disruption to virulence

Polymorphism in Gag Gene Cleavage Sites of HIV-1 Non-B Subtype and Virological Outcome of a First-Line Lopinavir/Ritonavir Single Drug Regimen

Virological failure on a boosted-protease inhibitor (PI/r) first-line triple combination is usually not associated with the detection of resistance mutations in the protease gene. Thus, other resistance pathways are being investigated. First-line PI/r monotherapy is the best model to investigate in vivo if the presence of mutations in the cleavage sites (CS) of gag gene prior to any antiretroviral treatment might influence PI/r efficacy. 83 patients were assigned to initiate antiretroviral treatment with first-line lopinavir/r monotherapy in the randomised Monark trial. We compared baseline sequence of gag CS between patients harbouring B or non-B HIV-1 subtype, and between those who achieved viral suppression and those who experienced virological failure while on LPV/r monotherapy up to Week 96. Baseline sequence of gag CS was available for 82/83 isolates; 81/82 carried at least one substitution in gag CS compared to HXB2 sequence. At baseline, non-B subtype isolates were significantly more likely to harbour mutations in gag CS than B subtype isolates (p<0.0001). Twenty-three patients experienced virological failure while on lopinavir/r monotherapy. The presence of more than two substitutions in p2/NC site at baseline significantly predicted virological failure (p = 0.0479), non-B subtype isolates being more likely to harbour more than two substitutions in this specific site. In conclusion, gag cleavage site was highly polymorphic in antiretroviral-naive patients harbouring a non-B HIV-1 strain. We show that pre-therapy mutations in gag cleavage site sequence were significantly associated with the virological outcome of a first-line LPV/r single drug regimen in the Monark trial

Occurrence of Corynebacterium striatum as an emerging antibiotic-resistant nosocomial pathogen in a Tunisian hospital

Corynebacterium striatum is a nosocomial opportunistic pathogen increasingly associated with a wide range of human infections and is often resistant to several antibiotics. We investigated the susceptibility of 63 C. striatum isolated at the Farhat-Hached hospital, Sousse (Tunisia), during the period 2011?2014, to a panel of 16 compounds belonging to the main clinically relevant classes of antimicrobial agents. All strains were susceptible to vancomycin, linezolid, and daptomycin. Amikacin and gentamicin also showed good activity (MICs90 = 1 and 2 mg/L, respectively). High rates of resistance to penicillin (82.5%), clindamycin (79.4%), cefotaxime (60.3%), erythromycin (47.6%), ciprofloxacin (36.5%), moxifloxacin (34.9%), and rifampicin (25.4%) were observed. Fifty-nine (93.7%) out of the 63 isolates showed resistance to at least one compound and 31 (49.2%) were multidrug-resistant. Twenty-nine resistance profiles were distinguished among the 59 resistant C. striatum. Most of the strains resistant to fluoroquinolones showed a double mutation leading to an amino acid change in positions 87 and 91 in the quinolone resistance-determining region of the gyrA gene. The 52 strains resistant to penicillin were positive for the gene bla, encoding a class A ?-lactamase. Twenty-two PFGE patterns were identified among the 63 C. striatum, indicating that some clones have spread within the hospital

Evidence for Induction of Integron-Based Antibiotic Resistance by the SOS Response in a Clinical Setting

Bacterial resistance to β-lactams may rely on acquired β-lactamases encoded by class 1 integron-borne genes. Rearrangement of integron cassette arrays is mediated by the integrase IntI1. It has been previously established that integrase expression can be activated by the SOS response in vitro, leading to speculation that this is an important clinical mechanism of acquiring resistance. Here we report the first in vivo evidence of the impact of SOS response activated by the antibiotic treatment given to a patient and its output in terms of resistance development. We identified a new mechanism of modulation of antibiotic resistance in integrons, based on the insertion of a genetic element, the gcuF1 cassette, upstream of the integron-borne cassette blaOXA-28 encoding an extended spectrum β-lactamase. This insertion creates the fused protein GCUF1-OXA-28 and modulates the transcription, the translation, and the secretion of the β-lactamase in a Pseudomonas aeruginosa isolate (S-Pae) susceptible to the third generation cephalosporin ceftazidime. We found that the metronidazole, not an anti-pseudomonal antibiotic given to the first patient infected with S-Pae, triggered the SOS response that subsequently activated the integrase IntI1 expression. This resulted in the rearrangement of the integron gene cassette array, through excision of the gcuF1 cassette, and the full expression the β-lactamase in an isolate (R-Pae) highly resistant to ceftazidime, which further spread to other patients within our hospital. Our results demonstrate that in human hosts, the antibiotic-induced SOS response in pathogens could play a pivotal role in adaptation process of the bacteria

Rapid and Sensitive Detection of Yersinia pestis Using Amplification of Plague Diagnostic Bacteriophages Monitored by Real-Time PCR

BACKGROUND: Yersinia pestis, the agent of plague, has caused many millions of human deaths and still poses a serious threat to global public health. Timely and reliable detection of such a dangerous pathogen is of critical importance. Lysis by specific bacteriophages remains an essential method of Y. pestis detection and plague diagnostics. METHODOLOGY/PRINCIPAL FINDINGS: The objective of this work was to develop an alternative to conventional phage lysis tests--a rapid and highly sensitive method of indirect detection of live Y. pestis cells based on quantitative real-time PCR (qPCR) monitoring of amplification of reporter Y. pestis-specific bacteriophages. Plague diagnostic phages phiA1122 and L-413C were shown to be highly effective diagnostic tools for the detection and identification of Y. pestis by using qPCR with primers specific for phage DNA. The template DNA extraction step that usually precedes qPCR was omitted. phiA1122-specific qPCR enabled the detection of an initial bacterial concentration of 10(3) CFU/ml (equivalent to as few as one Y. pestis cell per 1-microl sample) in four hours. L-413C-mediated detection of Y. pestis was less sensitive (up to 100 bacteria per sample) but more specific, and thus we propose parallel qPCR for the two phages as a rapid and reliable method of Y. pestis identification. Importantly, phiA1122 propagated in simulated clinical blood specimens containing EDTA and its titer rise was detected by both a standard plating test and qPCR. CONCLUSIONS/SIGNIFICANCE: Thus, we developed a novel assay for detection and identification of Y. pestis using amplification of specific phages monitored by qPCR. The method is simple, rapid, highly sensitive, and specific and allows the detection of only live bacteria