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1.  Pretransplant Fecal Carriage of Extended-Spectrum β-Lactamase–producing Enterobacteriaceae and Infection after Liver Transplant, France 
Emerging Infectious Diseases  2012;18(6):908-916.
Bacterial infection after liver transplant is fairly common, mostly because liver transplant patients are severely ill and the surgery is very complex. Adding to the seriousness of this situation is that some bacteria are resistant to many antimicrobial drugs. However, treating all infections as drug resistant would lead to even more drug resistance, so only patients at highest risk should receive the most powerful drugs. But who is at highest risk? A recent study in France screened fecal samples of liver transplant candidates and found that post-operative infections were most likely for those patients who already had certain bacteria in their feces before surgery. Thus, fecal screening for those multiresistant bacteria should be considered for all liver transplant candidates so that if post-operative infection develops, those at high risk can receive the most specific drugs right away.
PMCID: PMC3358139  PMID: 22607885
liver transplantation; extended-spectrum β-lactamase; infection; fecal carriage; Enterobacteriaceae; transplant; liver; France; carbapenems; antimicrobial resistance; bacteria; ESBL
2.  In Vitro Selection of ramR and soxR Mutants Overexpressing Efflux Systems by Fluoroquinolones as Well as Cefoxitin in Klebsiella pneumoniae▿ 
The relationship between efflux system overexpression and cross-resistance to cefoxitin, quinolones, and chloramphenicol has recently been reported in Klebsiella pneumoniae. In 3 previously published clinical isolates and 17 in vitro mutants selected with cefoxitin or fluoroquinolones, mutations in the potential regulator genes of the AcrAB efflux pump (acrR, ramR, ramA, marR, marA, soxR, soxS, and rob) were searched, and their impacts on efflux-related antibiotic cross-resistance were assessed. All mutants but 1, and 2 clinical isolates, overexpressed acrB. No mutation was detected in the regulator genes studied among the clinical isolates and 8 of the mutants. For the 9 remaining mutants, a mutation was found in the ramR gene in 8 of them and in the soxR gene in the last one, resulting in overexpression of ramA and soxS, respectively. Transformation of the ramR mutants and the soxR mutant with the wild-type ramR and soxR genes, respectively, abolished overexpression of acrB and ramA in the ramR mutants and of soxS in the soxR mutant, as well as antibiotic cross-resistance. Resistance due to efflux system overexpression was demonstrated for 4 new antibiotics: cefuroxime, cefotaxime, ceftazidime, and ertapenem. This study shows that the ramR and soxR genes control the expression of efflux systems in K. pneumoniae and suggests the existence of efflux pumps other than AcrAB and of other loci involved in the regulation of AcrAB expression.
PMCID: PMC3101381  PMID: 21464248
3.  Genetic Diversity and Virulence Profiles of Escherichia coli Isolates Causing Spontaneous Bacterial Peritonitis and Bacteremia in Patients with Cirrhosis▿  
Journal of Clinical Microbiology  2010;48(8):2709-2714.
Among patients with cirrhosis, infections caused by Escherichia coli organisms that translocate from the gut are a frequent and severe complication. One hundred ten E. coli isolates from 110 cirrhotic patients with spontaneous bacterial peritonitis and/or spontaneous bacteremia were characterized for their phylogenetic group and virulence genotype (34 extraintestinal virulence factor genes). Genetic relatedness was investigated by enterobacterial repetitive intergenic consensus sequence type 2 (ERIC-2) PCR typing and multilocus sequence typing. Phylogenetic groups A, B1, B2, and D accounted for 24%, 4%, 48%, and 24% of the population, respectively. Overall, 68 distinct ERIC-2 profiles were encountered. Eleven clonal groups, represented by multiple isolates (2 to 11) from the same sequence type (ST) or sequence type complex, were identified. These clonal groups accounted for 54 (49%) isolates overall. Membership in one of these clonal groups was more frequent among B2 isolates than non-B2 isolates (67% versus 32%, P < 0.001). The most frequent sequence types were ST95 (n = 13) and ST73 (n = 8), followed by the ST14 and ST10 complexes (n = 7). ST131 and ST69 were represented by three isolates each. Clonal group-associated isolates exhibited a greater prevalence of 11 virulence genes, including pap elements, than the other isolates. However, no association between clonal groups and host factors, type of infection, or mortality was observed. In conclusion, E. coli isolates causing spontaneous bacterial peritonitis and bacteremia in cirrhotic patients are genetically diverse. However, approximately half of the isolates belong to familiar clonal groups and exhibit extensive virulence profiles that may be associated with greater invasive potential.
PMCID: PMC2916625  PMID: 20519468
4.  Early Diagnosis of Extrapulmonary Tuberculosis by a New Procedure Combining Broth Culture and PCR▿  
Journal of Clinical Microbiology  2009;47(5):1452-1457.
The diagnosis of extrapulmonary tuberculosis is difficult because of the paucibacillary nature of these infections. We developed a culture-enhanced PCR assay combining a preliminary step of broth culture in BacT/Alert MP bottles with the subsequent detection of Mycobacterium tuberculosis using the GenoType Mycobacteria Direct test. First, the procedure was applied to 10-fold-diluted suspensions of M. tuberculosis prepared in vitro. These experiments showed that a 15-day incubation time was required to detect bacilli in the suspension, with the lowest inoculum size yielding a single colony on Lowenstein-Jensen slants. The efficacy of culture-enhanced PCR at day 15 was subsequently evaluated with 225 nonrespiratory specimens from 189 patients with suspected tuberculosis. All these specimens were smear negative, and 31 (13.8%) from 27 patients were culture positive. The result of culture-enhanced PCR at day 15 was consistent with final culture results in all specimens tested. Compared to culture results, the sensitivity, specificity, positive predictive value, and negative predictive value were 100%. Four patients with a negative culture and a negative PCR result were diagnosed as having tuberculosis on the basis of histological findings or therapeutic response. When using a positive diagnosis of tuberculosis as a gold standard, the sensitivity, specificity, positive predictive value, and negative predictive value were 88.6%, 100%, 100%, and 97.9%, respectively. These results indicate that culture-enhanced PCR is a highly sensitive and specific method for the early detection of M. tuberculosis in extrapulmonary specimens.
PMCID: PMC2681850  PMID: 19321729
5.  Efflux Pump, the Masked Side of ß-Lactam Resistance in Klebsiella pneumoniae Clinical Isolates 
PLoS ONE  2009;4(3):e4817.
β-lactamase production and porin decrease are the well-recognized mechanisms of acquired ß-lactam resistance in Klebsiella pneumoniae isolates. However, such mechanisms proved to be absent in K. pneumoniae isolates that are non susceptible to cefoxitin (FOX) and succeptible to amoxicillin+clavulanic acid in our hospital. Assessing the role of efflux pumps in this β-lactam phenotype was the aim of this study.
MICs of 9 β-lactams, including cloxacillin (CLX), and other antibiotic families were tested alone and with an efflux pump inhibitor (EPI), then with both CLX (subinhibitory concentrations) and EPI against 11 unique bacteremia K. pneumoniae isolates displaying the unusual phenotype, and 2 ATCC strains. CLX and EPI-dose dependent effects were studied on 4 representatives strains. CLX MICs significantly decreased when tested with EPI. A similar phenomenon was observed with piperacillin+tazobactam whereas MICs of the other β-lactams significantly decreased only in the presence of both EPI and CLX. Thus, FOX MICs decreased 128 fold in the K. pneumoniae isolates but also16 fold in ATCC strain. Restoration of FOX activity was CLX dose-dependent suggesting a competitive relationship between CLX and the other β-lactams with regard to their efflux. For chloramphenicol, erythromycin and nalidixic acid whose resistance was also due to efflux, adding CLX to EPI did not increase their activity suggesting differences between the efflux process of these molecules and that of β-lactams.
This is the first study demonstrating that efflux mechanism plays a key role in the β-lactam susceptibility of clinical isolates of K. pneumoniae. Such data clearly evidence that the involvement of efflux pumps in ß-lactam resistance is specially underestimated in clinical isolates.
PMCID: PMC2652100  PMID: 19279676
6.  Accuracy and Potential Usefulness of Triplex Real-Time PCR for Improving Antibiotic Treatment of Patients with Blood Cultures Showing Clustered Gram-Positive Cocci on Direct Smears▿  
Journal of Clinical Microbiology  2008;46(6):2045-2051.
Bacterial identification and antibiotic susceptibility testing currently require 48 h when a first blood culture (BC) is positive for clustered gram-positive cocci on direct smear examination (DSE). Meanwhile, antibiotic treatment is often inadequate, reducing the chances of effective treatment or creating unnecessary selective pressure. A new real-time PCR (RT-PCR) technique that differentiates Staphylococcus aureus from coagulase-negative staphylococci (CoNS) and detects methicillin resistance in 90 min in BC bottles could help solve these problems. BC bottles from 410 patients with gram-positive cocci on DSE were processed by current methods, and patients' treatments were prospectively recorded. The RT-PCR assay was performed on aliquots of these BCs, which had been kept frozen. For the 121 patients who had true bacteremia, we established whether the faster availability of RT-PCR results could have led to the initiation of treatments different from those actually given. RT-PCR sensitivity and specificity were 100% for differentiating between S. aureus and CoNS and detecting methicillin resistance with two manufacturers' BC bottles. For 31/86 (36%) of the S. aureus-infected patients and for 8/35 (23%) of the CoNS-infected patients who either had suboptimal or nonoptimal treatment or were untreated 48 h after positivity was detected, the early availability of RT-PCR results could have allowed more effective treatment. Unnecessary glycopeptide treatments could have been avoided for 28 additional patients. The use of RT-PCR would increase treatment effectiveness in patients with staphylococcal bacteremia and reduce the selective pressure created by glycopeptides.
PMCID: PMC2446825  PMID: 18417663
7.  Evaluation and Updating of the Osiris Expert System for Identification of Escherichia coli β-Lactam Resistance Phenotypes 
Journal of Clinical Microbiology  2005;43(4):1846-1850.
Osiris is a video zone size reader for disk diffusion tests that includes a built-in extended expert system (EES). We evaluated the efficacy of the Osiris EES for the identification of β-lactam susceptibility phenotypes of Escherichia coli isolates. Fifteen β-lactam agents and three β-lactam-β-lactamase inhibitor combinations were tested by the disk diffusion method against 50 E. coli strains with well-characterized resistance mechanisms. The strains were screened for the production of extended-spectrum β-lactamase (ESBL) by the double-disk synergy test using a disk of amoxicillin-clavulanic acid with disks of the extended-spectrum cephalosporins and aztreonam. Overall, the EES accurately identified the phenotype for 78% of the strains, indicated an inexact phenotype for 17% of the strains, and could not find a matching phenotype for the remaining 5% of the strains. The percentage of correct identification for each resistance mechanism was 100% for inhibitor-resistant TEM and for TEM plus cephalosporinase, 88.9% for TEM and for ESBL, 70.8% for cephalosporinase overproduction, and 25% for oxacillinase. The main cause of discrepancy was the misidentification of oxacillinase as inhibitor-resistant TEM. The conventional double-disk synergy test failed to detect ESBL production in two strains (one producing VEB-1 and one producing CTX-M-14), but synergy between cefepime and amoxicillin-clavulanic acid was visible after the distance between the disks was reduced to 20 mm. After the interpretative guidelines of the EES were updated according to our results, the percentage of correct phenotype identification increased from 78 to 96%.
PMCID: PMC1081361  PMID: 15815007
8.  Prevalence, Molecular Epidemiology, and Clinical Significance of Heterogeneous Glycopeptide-Intermediate Staphylococcus aureus in Liver Transplant Recipients 
Journal of Clinical Microbiology  2003;41(11):5147-5152.
We investigated the prevalence, molecular epidemiology, and clinical significance of heterogeneous glycopeptide-intermediate Staphylococcus aureus (hGISA) isolates in 48 liver transplant recipients infected or colonized with methicillin-resistant S. aureus over a 5-year period. Strains were screened for hGISA on Mueller-Hinton agar containing 5 mg of teicoplanin per liter. Heterogeneous glycopeptide resistance was confirmed by the E-test method with a dense inoculum and a simplified method of population analysis. hGISA strains were found in 13 (27%) of the 48 patients studied. Eleven of the 13 strains shared a common multiresistant phenotype with homogeneous methicillin resistance and gentamicin resistance, and they were closely related according to the results of pulsed-field gel electrophoresis. Only 2 of the 13 patients infected or colonized with hGISA strains had previously received glycopeptide therapy. Most patients were successfully treated with vancomycin, but one patient who failed to respond to vancomycin subsequently died. These results suggest that the high prevalence of hGISA among our patients was due to the clonal spread of a multiresistant strain.
PMCID: PMC262463  PMID: 14605151
9.  Evaluation of the Osiris Expert System for Identification of β-Lactam Phenotypes in Isolates of Pseudomonas aeruginosa 
Journal of Clinical Microbiology  2003;41(8):3712-3718.
Osiris is a video zone size reader for disk diffusion tests featuring a built-in extended expert system (EES). The efficacy of the EES for the identification of the β-lactam susceptibility phenotypes of Pseudomonas aeruginosa isolates was evaluated. Thirteen β-lactams were tested in four laboratories by the disk diffusion test with 53 strains with well-characterized resistance mechanisms, including the production of 12 extended-spectrum β-lactamases (ESBLs). The plates were read with the Osiris system and the results were interpreted with the ESS, and then the phenotype identified by the EES was compared to the resistance mechanism. The strains were also screened for the presence of ESBL production by a double-disk synergy test by placing the strains between an extended-spectrum cephalosporin-containing disk and a clavulanic acid-containing disk at distances of 30, 20, 15, and 10 mm from each other. Overall, the EES accurately identified the phenotypes of 88.2% of the strains and indicated an association with several mechanisms for 3.8% of the strains. No phenotype was identified in four strains with low levels of penicillinase production. Misidentifications were observed for two penicillinase-producing strains: one strain with partially derepressed cephalosporinase production and one strain overexpressing the MexA-MexB-OprM efflux system. The production of only four ESBLs was detected by the standard synergy test with a 30-mm distance between the disks. The production of five further ESBLs was identified by reducing the distance to 20 mm, and the production of the last three ESBLs was detected only at a distance of 15 or 10 mm. Our results indicate that the Osiris EES is an effective tool for the identification of P. aeruginosa β-lactam phenotypes. A specific double-disk synergy test with reduced disk distances is necessary for the detection of ESBL production by this organism.
PMCID: PMC179830  PMID: 12904380

Results 1-9 (9)