Detection of a bla(SHV) extended-spectrum {beta}-lactamase in Salmonella enterica serovar Newport MDR-AmpC

Salmonella enterica serovar Newport MDR-AmpC expressing TEM-1b and extended-spectrum beta-lactamase SHV-12 was isolated from affected animals during an outbreak of salmonellosis that led to a 3-month closure of one of the largest equine hospitals in the United States.     

Outbreak caused by a multidrug-resistant Klebsiella pneumoniae clone carrying blaVIM-12 in a university hospital

From November 2006 to April 2007, nine nonrepetitive isolates of Klebsiella pneumoniae with reduced susceptibility or resistance to carbapenems were recovered from clinical specimens from separate patients hospitalized in a tertiary care hospital. The imipenem-EDTA synergy test was positive for all isolates. PCR, sequencing, and transferability experiments revealed the novel bla(VIM-12) metallo-beta-lactamase gene, which was plasmid mediated and located in a class 1 integron. Pulsed-field gel electrophoresis demonstrated a single macrorestriction pattern, indicating the clonal spread of VIM-12-producing K. pneumoniae.     

Primary liver abscess due to CC23-K1 virulent clone of Klebsiella pneumoniae in France

Since the mid-1980s, Klebsiella pneumoniae hypermucoviscous isolates have emerged in Taiwan and other Asian countries. We reported the first autochthonous European liver abscess due to an ST57 isolate, which belongs to virulent clonal complex CC23-KI. This case highlights the emergence in France and Europe of hypermucoviscous virulent K. pneumoniae isolates.     

Non-random employment of V{beta}6 and J{beta} gene elements and conserved amino acid usage profiles in CDR3 regions of human fetal and adult TCR {beta} chain rearrangements

We have studied the usage of V_ß36, D_ß, andJ_ß elements, and the composition of the CDR3 regionsof human fetal TCR ß chain rearrangements In a 17week old fetal thymus and In fetal liver, bone marrow, spleen,and cord blood at 11 and 13 weeks of gestation. These fetalsequences were compared with TCR ß chain rearrangementsobtained from post-partum thymus, adult spleen, and adult peripheralblood mononuclear cells. Both fetal and adult TCR V_ß6rearrangements exhibited a non-random usage of V_ßand J_ß elements. Up to 90% of the sequences obtainedat 11 weeks of gestation used J_ß2 elements, most notablyJ_ß2.1. In the 13 and 17 week old fetal and in theadult tissues, J_ß1 elements were used In 30% of therearrangements while, within the J_ß2 rearrangements,J_ß2.1 and J_ß2.7 were used most frequently.Both fetal and adult TCR ß chain CDR3 regions showednon-random usage of amlno acids. However, the early fetal repertoirewas further limited due to the relative absence of N-reglonsin up to 60% of the 11 and 13 week old TCR ß chainrearrangements, resulting In smaller antigen binding sites.In fetal and adult TCR ß chain rearrangements thedistribution patterns of the length of N-regions and the usageprofiles of J_ß elements were similar in hematopoletlcand peripheral organs, suggesting no apparent preference forparticular TCR ß chain rearrangements. On the whole,the data showed that both the available fetal and adult TCRV_ß6 repertoires are seemingly smaller than expectedon the basis of random usage of V_ß and J_ßelements and amlno acid composition of the CDR3 regions.     

A new mutagenicity assay method for frameshift mutagens based on deleting or inserting a guanosine nucleotide in the {beta}-lactamase gene

The conventional method of site-directed mutagenesis was usedto develop two Salmonella strains, JK-1 and JK-2, for detectingframeshift mutagens. The JK-1 strain was derived from Salmonellatyphimurium TA1537 strain transformed by a mutant construct.A guanosine nucleotide was inserted between nucleotide residues312 and 313 of the ß-lactamase gene. The JK-2 strainwas obtained by the same procedure, but a guanosine nucleotidein position 315 of the ß-lactamase gene was deleted.The strains were tested with ten frameshift mutagens and therevertants were selected by ampicillin resistance. Representativemutagens including 2-nitrofluorene (2-NF), 2-acetylaminofluorene(AAF), 9-aminoacridine (9-AA), 2,7-diaminofluorene (2,7-DAF)and 2-methoxy-6-chloro-9-(3-(ethyl-2-chloro-ethyl)-aminopropylamino)acridine(ICR-170) were more potent in the JK-1 strain than the JK-2strain, and the number of revertant colonies were dose related.Under the same conditions, the ampicillin test was more sensitivethan the Ames test. Other types of compounds such as 2-methoxy-6-chloro-9-(2-chloroethylaminopropylamino)acridine(ICR-191), benzo[     

bla KPC and rmtB on a single plasmid in Enterobacter amnigenus and Klebsiella pneumoniae isolates from the same patient

Enterobacter amnigenus (EA76) and Klebsiella pneumoniae (KP76) isolates with multidrug-resistant (MDR) patterns were identified from the same patient in the neurosurgery department of our hospital. An outbreak of MDR K. pneumoniae had also occurred in this department. To characterize the resistance mechanism and molecular epidemiology of these isolates, sequential experiments including antimicrobial susceptibility testing, polymerase chain reaction (PCR), plasmid analysis, pulsed field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) were performed. EA76 and KP76 were resistant to all of the antibiotics tested, except colistin and tigecycline. blaKPC-2, blaTEM-1, blaSHV-12, blaCTX-M-3, blaCTX-M-14, and rmtB genes were identified in both isolates, with blaKPC-2, blaTEM-1, blaCTX-M-14, and rmtB being co-carried on one plasmid in each isolate. Further analysis showed different restriction patterns between the two KPC-carrying plasmids. Of the 11 carbapenem-resistant isolates found in the outbreak, all were resistant to all of the β-lactams tested, with 63.64% (7/11) also exhibiting resistance to aminoglycosides and 72.73% (8/11) exhibiting resistance to quinolones. PCR analysis and molecular typing of the 11 K. pneumoniae strains revealed that the seven aminoglycoside-resistant isolates shared the same antibiotic-resistant gene pattern and identical or one-band-difference PFGE profiles relative to KP76. In addition, all of the eight aminoglycoside-resistant isolates, including KP76, belonged to the national epidemic clone ST11. The overall results indicate the emergence of E. amnigenus and outbreak of ST11 K. pneumoniae, with both co-harboring blaKPC and rmtB genes on a single plasmid in our neurosurgery wards.     

Functional expression of a human TCR{beta} gene in transgenic mice

A functionally rearranged TCRß (Tcrb) gene was isolatedfrom a cloned human T helper cell recognizing the CS.T3 epitopeof Plasmodium falciparum with HLA-DR2. Transgenic mice weregenerated by co-injection of the human gene together with themouse Tcrb enhancer. Analysis of transgenic mice shows thatthe functional Tcrb gene of xenogenic, i.e. human, origin exertsallelic exclusion of endogenous Tcrb genes. Cytofluorometricanalysis revealed expression of the human TCRß chainon virtually all thymocytes and peripheral T cells togetherwith endogenous TCRß chains and CD3 components. Nosurface expression of mouse TCRß chain or rearrangementof endogenous Tcr genes was detectable. Expression of the hybridreceptor causes a reduction in the number of thymocytes anda bias for CD4⁺CD8^– T cells in the thymus as comparedwith non-transgenic littermates. Peripheral transgenic T cellsmount a normal prollferative response against allogenelc targetsin mixed lymphocyte reactions. These results show that a hybridmouse/human TCR is able to pass positive and negative selectionin the thymus, and is functional in transgenic mice.     

A reverse mutagenicity assay for alkylating agents based on a point mutation in the {beta}-lactamase gene at the active site serine codon

The serine at the active site of ß-lactamase is responsiblefor the ester link to the acyl group of ß-lactam duringhydrolysis of the substrate to its acid derivatives. A constructwas made from a plasmid in which the active-site serine of ß-lactamasewas substituted by glycine by site-directed mutation. This mutationresults in the loss of ß-lactamase activity. Thisplasmid was used to transform Salmonella typhimurium TA1535.When the new strain JK947 was treated with a mutagen such asN-methyl-N'-nitro-nitrosoguanidine (MNNG), the bacteria couldbe recovered as they became ampicillin resistant. The sequenceof the active-site serine codon in these revertants was mutatedfrom GGC to AGC. Based upon these findings, we developed a modelreverse mutagenicity assay. In this procedure, we treated JK947with a test chemical, such as N-methyl-nitrosourea (MNU), dimethylsulphate (DMS) or methylmethane sulphonate (MMS), for 30 min,and then scored the revertants on agar plates containing 50µ g/ ml ampicillin after incubation at 37°C for 16h. MNU and MNNG were more potent than DMS and MMS in this assay.Treatment with MNU and MNNG resulted in larger colony numbersin our test than in the Ames test. However, our test was lesssensitive to DMS and MMS than the Ames test. ¹To whom correspondence should be addressed     

Recurring Klebsiella pneumoniae pyogenic liver abscesses in a resident of San Diego, California, due to a K1 strain carrying the virulence plasmid

We report a diabetic patient who had three episodes of cryptogenic liver abscess due to Klebsiella pneumoniae. He was a Caucasian who lived in California and had no epidemiological connection to East Asia. The isolate from his third episode was a hyperviscous K1 strain that carried the Klebsiella virulence plasmid.     

Violation of allelic exclusion of the T cell receptor {beta} genes in a helper T cell clone

The 4-hydroxy-3-nltrophenylacetyl coupled to chicken gamma globulinspecific and I-A^b restricted helper T cell clone IH4 carriesa V_{ß8 1}–D_ß2.1–J_ß2.3-rearrangementon one and a V_ß16–D_ß2.1–J_ß2.5-rearrangementon the other chromosome. Both rearranged ß genes aretranscribed and the products of both genes are expressed onthe cell surface. This result implicates the absence of allelicexclusion of the T cell receptor ß genes in this Tcell clone. The finding of a pseudoleader 5' to the functionalleader exon of the V_ß16 gene suggests that this geneis not efficiently translated and that the amount of V_ß16chains synthesized is not sufficient to activate the inhibitorymechanism preventing a second V_ß to D_ßJ_ßrearrangement.     

Carbapenem Heteroresistance in VIM-1-Producing Klebsiella pneumoniae Isolates Belonging to the Same Clone: Consequences for Routine Susceptibility Testing

Susceptibility results with low reproducibility by the same or different methods have been observed for metallo-beta-lactamase (MBL)-producing Enterobacteriaceae. Eighteen VIM-1-producing Klebsiella pneumoniae isolates (one per patient) belonging to a single epidemic clone in our hospital (2005 to 2008) but with different susceptibilities to carbapenems were studied. Imipenem MICs ranged from 8 to >128 mg/liter by standard CLSI microdilution, from ≤1 to >8 mg/liter by the semiautomatic Wider system, and from 0.75 to >32 mg/liter by Etest. Meropenem MICs ranged from 0.5 to 128, ≤1 to >8, and 0.38 to >32 mg/liter, respectively. Ertapenem MICs by CLSI microdilution and Etest ranged from 1 to 64 and 0.75 to >32 mg/liter, respectively. The rates of essential agreement (±1 log₂ dilution) for imipenem and meropenem MICs between the Wider system and the reference microdilution method were 45% and 49%, respectively. Those between Etest and the reference microdilution method for imipenem, meropenem, and ertapenem MICs were 33%, 67%, and 84%. The rates of very major errors for the Wider system and Etest were 33% and 28% for imipenem and 25% and 75% for meropenem, respectively. Low MIC reproducibility was observed even when the same inoculum was used (differences up to 4-fold dilutions). Heteroresistance was suspected due to the presence of colonies in the Etest inhibition zone. It was confirmed by population analysis profiles of 4 isolates displaying different imipenem MICs, with the exception of an OmpK36-porin-deficient isolate that homogeneously expressed carbapenem resistance (MIC, >128 mg/liter). Low carbapenem MIC reproducibility could be due to the presence of resistant subpopulations and variable expression of the resistance mechanisms. Since carbapenem MICs are not good markers of MBL production, reliable and reproducible phenotypic methods are needed to detect the presence of this mechanism.Resistance to carbapenems due to metallo-beta-lactamases (MBLs) in Enterobacteriaceae is increasingly recognized, and different levels of resistance to carbapenems have been observed in these isolates (18). The detection of MBL-carrying isolates in clinical laboratories presents practical difficulties when routine susceptibility testing is performed (10, 28). Automated systems do not accurately detect enzyme-mediated carbapenem resistance when it is expressed at low levels, and MBL-carrying organisms can even appear to be fully susceptible to these compounds (9, 28). Additionally, reproducible results within the same method or among different methods, such as disk diffusion or Etest, are not always obtained, and discrepancies in the susceptibility to imipenem and meropenem of carbapenemase (KPC or VIM types)-producing Klebsiella pneumoniae isolates have already been reported (10, 28).In addition, some studies describing heteroresistance to carbapenems in Acinetobacter baumannii isolates demonstrate its presence by the growth of colonies in the inhibition zone of the Etest strip (12, 19). This phenomenon has also been reported for Klebsiella pneumoniae (28), Pseudomonas aeruginosa (20), and, more recently, Enterobacter aerogenes isolates (11).The objective of this work was to analyze the carbapenem susceptibility profiles of VIM-1-producing K. pneumoniae isolates by different susceptibility testing methods. These isolates belong to a single clone that was involved in an epidemic in our hospital (2005 to 2008) affecting 18 patients in different wards (25).     

Early detection of colonization by VIM-1-producing Klebsiella pneumoniae and NDM-1-producing Escherichia coli in two children returning to France

Rapid identification of metallo-β-lactamase-producing Gram-negative species is crucial for the timely implementation of infection control measures. We describe two pediatric cases in which colonization by VIM-1- and New Delhi metallo-beta-lactamase 1-producing Enterobacteriaceae was rapidly detected by phenotypic and genotypic methods. Phenotypic methods can be useful for routine detection of carbapenemase production.     

Meningoencephalitis due to community Klebsiella pneumoniae in an adult immunocompetent: a case report

We report the first Moroccan case of community-acquired meningoencephalitis due to Klebsiella pneumoniae in an adult immunocompetent without medical history, complicated of cerebral vasculitis and right endophthalmitis. K. pneumoniae is exceptionally responsible for meningitis community in the world excepted in some countries of Southeast Asia. It usually occurs on fragile field and is associated with high mortality. The early management of the patient, the immunocompetent field and the wild phenotype of the isolated strain probably allowed to get a medical cure with some sequelae.     

Bacteremia due to extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae in a pediatric oncology ward: clinical features and identification of different plasmids carrying both SHV-5 and TEM-1 genes

Thirteen patients who had 16 episodes of bacteremia were observed between 1993 and 1997 in a pediatric oncology ward with a high background isolation rate of cefotaxime- or aztreonam-resistant gram-negative bacteria. Four blood isolates were Escherichia coli and 12 were Klebsiella pneumoniae, and these isolates harbored extended-spectrum beta-lactamases (ESBLs). All episodes of bacteremia were nosocomial, all except one of the episodes occurred in neutropenic patients, and all patients were treated with piperacillin or ceftazidime with amikacin and cefazolin prior to the onset of bacteremia. Nine of 13 patients were receiving extended-spectrum beta-lactam treatment when the bacteremias caused by ESBL producers occurred. Molecular studies revealed that four K. pneumoniae SHV-2-producing isolates from 1994 were of the same clone. Other ESBL producers, including six that carried both TEM-1 and SHV-5, five that carried SHV-5, and one that carried SHV-2 alone, were unrelated. In conclusion, SHV-5 was present in 11 of the 16 isolates and coexisted with TEM-1 in 6 isolates. Acquisition of resistance genes probably occurred under antibiotic selection pressure. This study highlights the importance of routine checks for and detection of ESBL producers. Effective therapy against ESBL producers should be considered early for children who have malignancies and neutropenia and who are septic, despite treatment with a regimen that includes an extended-spectrum beta-lactam, in a clinical setting of an increased incidence of ESBL-producing bacteria.     

Role of the CDR1 region of the TCR {beta} chain in the binding to purified MHC-peptide complex

Single alanine substitutions were introduced into the CDR1 regionof the ß chain of a K^d-restricted TCR. Mutants andwild-type TCR were attached to the chain of the CD3 complexand expressed at the surface of a rat basophil cell line. Transfectantswere tested for the binding of purified soluble K^d-peptlde complexes.With this experimental system, accessory molecules are unlikelyto play a major role and the contribution of each residue tothe interaction can be addressed. Results show that all positionsin the CDR1 region are involved in the binding to the K^d-peptidecomplex but at varying degrees. These effects are discussedin relation to a molecular model of the TCR. Comparison of theseresults with previous data obtained in a T cell hybridoma systemsuggests the existence of a threshold in the TCR affinity necessaryfor mature T cell activation.