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1.  Abrupt Emergence of a Single Dominant Multidrug-Resistant Strain of Escherichia coli 
The Journal of Infectious Diseases  2013;207(6):919-928.
Background. Fluoroquinolone-resistant Escherichia coli are increasingly prevalent. Their clonal origins—potentially critical for control efforts—remain undefined.
Methods. Antimicrobial resistance profiles and fine clonal structure were determined for 236 diverse-source historical (1967–2009) E. coli isolates representing sequence type ST131 and 853 recent (2010–2011) consecutive E. coli isolates from 5 clinical laboratories in Seattle, Washington, and Minneapolis, Minnesota. Clonal structure was resolved based on fimH sequence (fimbrial adhesin gene: H subclone assignments), multilocus sequence typing, gyrA and parC sequence (fluoroquinolone resistance-determining loci), and pulsed-field gel electrophoresis.
Results. Of the recent fluoroquinolone-resistant clinical isolates, 52% represented a single ST131 subclonal lineage, H30, which expanded abruptly after 2000. This subclone had a unique and conserved gyrA/parC allele combination, supporting its tight clonality. Unlike other ST131 subclones, H30 was significantly associated with fluoroquinolone resistance and was the most prevalent subclone among current E. coli clinical isolates, overall (10.4%) and within every resistance category (11%–52%).
Conclusions. Most current fluoroquinolone-resistant E. coli clinical isolates, and the largest share of multidrug-resistant isolates, represent a highly clonal subgroup that likely originated from a single rapidly expanded and disseminated ST131 strain. Focused attention to this strain will be required to control the fluoroquinolone and multidrug-resistant E. coli epidemic.
doi:10.1093/infdis/jis933
PMCID: PMC3571447  PMID: 23288927
Escherichia coli infections; antimicrobial resistance; extended-spectrum β-lactamase; CTX-M-15; fluoroquinolone resistance; multidrug resistance; sequence type ST131; multilocus sequence typing; molecular epidemiology; FimH
2.  Different Factors Associated with CTX-M-Producing ST131 and Non-ST131 Escherichia coli Clinical Isolates 
PLoS ONE  2013;8(9):e72191.
Objectives
To determine factors associated with CTX-M-producing ST131 Escherichia coli which is the worldwide predominant lineage among CTX-M-producing E. coli isolates.
Methods
Consecutive inpatients with a clinical sample positive for CTX-M-producing E. coli and considered as cases in a previous 8-month (2008–2009) case-control study performed in ten university hospitals in the Paris area were included in the present sub-population study. Patients with a CTX-M-producing ST131 E. coli clinical isolate were compared with those with a CTX-M-producing non-ST131 E. coli clinical isolate with regard to 66 variables. Variables were first compared using univariate logistic regression, then a multivariate analysis using a backward selection with variables with p-value <0.1 in univariate analysis was carried out.
Results
Fifty-five patients with a CTX-M-producing ST131 E. coli clinical isolate were compared to 97 patients with a CTX-producing non-ST131 E. coli clinical isolate. Multivariate analysis showed that only previous residence in long term care facilities (OR = 4.4; 95% CI = 1.3–14.7) was positively associated with a CTX-M-producing ST131 E. coli isolate. However, it also showed that regular consumption of poultry products (OR = 0.2; 95% CI = 0.1–0.6), having had at least one device in the preceding 6 months (OR = 0.3; 95% CI = 0.1–0.7) and stay in ICU (OR = 0.2; 95% CI = 0.05–0.8) were negatively associated with isolation of CTX-M-producing ST131 E. coli from clinical samples.
Conclusions
This study provides more insight into the epidemiological features of ST131 and non-ST131 E. coli producing CTX-M enzymes. It shows, for the first time, that isolation of CTX-M-producing ST131 E. coli from clinical samples is not linked to consumption of various foods and confirms that residence in long term care facilities is a predictor of these isolates.
doi:10.1371/journal.pone.0072191
PMCID: PMC3762868  PMID: 24023730
4.  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.
doi:10.3201/eid1806.110139
PMCID: PMC3358139  PMID: 22607885
liver transplantation; extended-spectrum β-lactamase; infection; fecal carriage; Enterobacteriaceae; transplant; liver; France; carbapenems; antimicrobial resistance; bacteria; ESBL
5.  Comparison of Escherichia coli ST131 Pulsotypes, by Epidemiologic Traits, 1967–2009 
Emerging Infectious Diseases  2012;18(4):598-607.
Certain high-prevalence pulsed-field gel electrophoresis types exhibited distinctive temporal patterns and epidemiologic associations.
Escherichia coli sequence type 131 (ST131), an emerging disseminated public health threat, causes multidrug-resistant extraintestinal infections. Among 579 diverse E. coli ST131 isolates from 1967–2009, we compared pulsotypes (>94% similar XbaI pulsed-field gel electrophoresis profiles) by collection year, geographic origin, source, and antimicrobial drug–resistance traits. Of 170 pulsotypes, 65 had >2 isolates and accounted for 85% of isolates. Although extensively dispersed geographically, pulsotypes were significantly source specific (e.g., had little commonality between humans vs. foods and food animals). The most prevalent pulsotypes were associated with recent isolation, humans, and antimicrobial drug resistance. Predominant pulsotype 968 was associated specifically with fluoroquinolone resistance but not with extended-spectrum β-lactamase production or blaCTX-M-15. Thus, several highly successful antimicrobial drug–resistant lineages within E. coli ST131 have recently emerged and diffused extensively among locales while maintaining a comparatively restricted host/source range. Identification of factors contributing to this behavior of ST131 could help protect public health.
doi:10.3201/eid1804.111627
PMCID: PMC3309662  PMID: 22469129
Escherichia coli infections; ST131; sequence type 131; CTX-M-15; fluoroquinolone resistance; antimicrobial resistance; extended-spectrum beta-lactamases; pulsed-field gel electrophoresis; bacteria; sequence typing
6.  Patient's Origin and Lifestyle Associated with CTX-M-Producing Escherichia coli: A Case-Control-Control Study 
PLoS ONE  2012;7(1):e30498.
Background
Global dissemination of Escherichia coli producing CTX-M extended-spectrum β-lactamases (ESBL) is a public health concern. The aim of the study was to determine factors associated with CTX-M- producing E. coli infections among patients hospitalised in the Assistance Publique-Hôpitaux de Paris, the largest hospital system in France (23 000 beds), through a prospective case-control-control study.
Methods/Principal Findings
From November 2008 to June 2009, 152 inpatients with a clinical sample positive for CTX-M-producing E. coli (cases), 152 inpatients with a clinical sample positive for non ESBL-producing E. coli on the day or within the three days following case detection (controls C1), and 152 inpatients with culture-negative clinical samples since the beginning of hospitalisation and until three days after case detection (controls C2) were included in ten hospitals of the Paris area. Factors studied were related to patient's origin, lifestyle and medical history as well as care during hospitalisation. Those independently associated with CTX-M-producing E. coli were determined. Three independent factors were common to the two case-control comparisons: birth outside of Europe (cases vs C1: OR1 = 2.4; 95%CI = [1.3–4.5] and cases vs C2: OR2 = 3.1; 95%CI = [1.4–7.0]), chronic infections (OR1 = 2.9; 95%CI = [1.3–6.9] and OR2 = 8.7; 95%CI = [2.0–39.7]), and antibiotic treatment between hospital admission and inclusion (OR1 = 2.0; 95%CI = [1.0–3.8] and OR2 = 3.3; 95%CI = [1.5–7.2]). Cases were also more likely to be (i) functionally dependent before hospitalisation than C2 (OR2 = 7.0; 95%CI = [2.1–23.5]) and (ii) living in collective housing before hospitalisation than C2 (OR2 = 15.2; 95%CI = [1.8–130.7]) when CTX-M-producing E. coli was present at admission.
Conclusion
For the first time, patient's origin and lifestyle were demonstrated to be independently associated with isolation of CTX-M-producing E. coli, in addition to health care-related factors.
doi:10.1371/journal.pone.0030498
PMCID: PMC3267726  PMID: 22299043
7.  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.
doi:10.1128/AAC.00156-11
PMCID: PMC3101381  PMID: 21464248
8.  Membrane Efflux and Influx Modulate both Multidrug Resistance and Virulence of Klebsiella pneumoniae in a Caenorhabditis elegans Model▿  
Antimicrobial Agents and Chemotherapy  2010;54(10):4373-4378.
Cross-resistance to cefoxitin (FOX), chloramphenicol (CMP), and quinolones (nalidixic acid [NAL]) related to a putative efflux system overexpression has recently been reported for Klebsiella pneumoniae. The potential impact of this multidrug resistance (MDR) on the virulence of K. pneumoniae was evaluated in the Caenorhabditis elegans model. For 2 of the 3 MDR clinical isolates studied, a significant increase in acrB transcription was found in comparison with their antibiotic-susceptible revertants. ATCC 138821 and MDR, revertant, and derivative strains with altered porin expression were studied. Strains proved or suspected to overexpress an efflux system were significantly more virulent than the ATCC and revertant strains (time to kill 50% of nematodes [LT50] in days: 3.4 to 3.8 ± 0.2 versus 4.1 to 4.4 ± 0.3, P < 0.001). Inversely, strains with altered porin expression were significantly less virulent, independently of the expression level of efflux system (LT50 = 5.4 to 5.6 ± 0.2, P < 0.001). Altered porin expression did not change MICs of CMP and NAL but did those of FOX (4 to 16× MIC) and ertapenem (16 to 64× MIC). The strains with a normally or an overexpressed efflux system that received the β-lactamase CTX-M-15 became more widely resistant without modification of their virulence potential, suggesting that balance between resistance and virulence is dependent on the type of resistance mechanisms. In conclusion, this study shows that the expression of both efflux systems and porins is a key factor not only for antibiotic resistance but also virulence potential in K. pneumoniae.
doi:10.1128/AAC.01607-09
PMCID: PMC2944602  PMID: 20679507
9.  Broad-Range 16S rRNA PCR with Cerebrospinal Fluid May Be Unreliable for Management of Postoperative Aseptic Meningitis ▿  
Journal of Clinical Microbiology  2010;48(9):3331-3333.
We previously demonstrated that discontinuing presumptive antibiotic treatment in cases of negative conventional cultures is safe and effective for patients with postoperative aseptic meningitis (PAM). Here, we prospectively investigated 32 patients with postoperative meningitis. All 26 patients with PAM diagnosed on the basis of conventional cultures demonstrated negative 16S rRNA PCR results. Our results suggest that the PCR technique does not change PAM management.
doi:10.1128/JCM.00008-10
PMCID: PMC2937738  PMID: 20592153
10.  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.
doi:10.1128/JCM.00516-10
PMCID: PMC2916625  PMID: 20519468
11.  Similarity and Divergence among Adherent-Invasive Escherichia coli and Extraintestinal Pathogenic E. coli Strains ▿  
Journal of Clinical Microbiology  2009;47(12):3968-3979.
Adherent-invasive Escherichia coli (AIEC) pathovar strains, which are associated with Crohn's disease, share many genetic and phenotypic features with extraintestinal pathogenic E. coli (ExPEC) strains, but little is known about the level of genetic similarity between the two pathovars. We aimed to determine the frequency of strains with the “AIEC phenotype” among a collection of ExPEC strains and to further search for a common phylogenetic origin for the intestinal and extraintestinal AIEC strains. The adhesion, invasion, and intramacrophage replication capabilities (AIEC phenotype) of 63 ExPEC strains were determined. Correlations between virulence genotype and AIEC phenotype and between intestinal/extraintestinal origin, serotype, and phylogroup were evaluated for the 63 ExPEC and 23 intestinal AIEC strains. Phylogenetic relationships between extraintestinal and intestinal AIEC strains were determined using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis. Only four (6.35%) ExPEC strains, belonging to the O6:H1, O83:H1, and O25:H4 serotypes, were classified as having an AIEC phenotype. These strains were found to be genetically related to some intestinal AIEC strains of the same serotypes as revealed by MLST. No particular virulence gene sets correlated with the intestinal/extraintestinal origin of the strains or with the AIEC phenotype, whereas the gene sets did correlate with the serogroup. We identified two intestinal AIEC strains and one extraintestinal AIEC strain belonging to the O25:H4 serotype that also belonged to the emerging and virulent clonal group ST131. In conclusion, the ExPEC and AIEC pathovars share similar virulence gene sets, and certain strains are phylogenetically related. However, the majority of ExPEC strains did not behave like AIEC strains, thus confirming that the AIEC pathovar possesses virulence-specific features that, to date, are detectable only phenotypically.
doi:10.1128/JCM.01484-09
PMCID: PMC2786640  PMID: 19828750
12.  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.
doi:10.1128/JCM.00066-09
PMCID: PMC2681850  PMID: 19321729
13.  Absence of CTX-M Enzymes but High Prevalence of Clones, Including Clone ST131, among Fecal Escherichia coli Isolates from Healthy Subjects Living in the Area of Paris, France▿  
Journal of Clinical Microbiology  2008;46(12):3900-3905.
Quinolone-resistant and CTX-M-15-producing Escherichia coli isolates belonging to clone ST131 have been reported in the community. This study was designed to identify these E. coli isolates in the stools of 332 independent healthy subjects living in the area of Paris, France. Stools were plated on media without antibiotics, in order to obtain the dominant (Dm) fecal E. coli strain, and with nalidixic acid (NAL) and cefotaxime. Quinolone susceptibility, phylogenetic groups, and molecular profiles, including multilocus sequence types (ST), were determined for all NAL-resistant (NAL-R) isolates. Groups were also determined for the Dm strains from participants with NAL-R isolates and from a subgroup without NAL-R isolates. All B2 isolates were typed; pulsed-field gel electrophoresis was performed for the ST131 isolates, and the results were compared with those for intercontinental clone ST131. Two participants (0.6%) had extended-spectrum β-lactamase-producing (SHV-2, TEM-52) fecal E. coli isolates, and 51 (15%) had NAL-R isolates; 51% of NAL-R isolates belonged to phylogenetic group A, 31% to group D, 16% to group B2, and 2% to group B1. The Dm strain was NAL-R in 3.3% of the 332 subjects. Forty-nine percent of the NAL-R isolates belonged to clones: ST10 and ST606 for group A isolates, ST117 and ST393 for group D isolates. Of all B2 isolates studied from 100 subjects (8 NAL-R strains; 19 NAL-susceptible dominant strains), 52% belonged to three clones: ST131 (n = 7), ST95 (n = 4), and ST141 (n = 3). This is the first study to show the presence of fecal E. coli isolates of clone ST131 in 7% of independent healthy subjects not colonized by CTX-M-15-producing isolates.
doi:10.1128/JCM.00734-08
PMCID: PMC2593250  PMID: 18842941
14.  Efflux Pump, the Masked Side of ß-Lactam Resistance in Klebsiella pneumoniae Clinical Isolates 
PLoS ONE  2009;4(3):e4817.
Background
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.
Methodology/Findings
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.
Conclusion
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.
doi:10.1371/journal.pone.0004817
PMCID: PMC2652100  PMID: 19279676
15.  Virulent Synergistic Effect between Enterococcus faecalis and Escherichia coli Assayed by Using the Caenorhabditis elegans Model 
PLoS ONE  2008;3(10):e3370.
Background
The role of enterococci in the pathogenesis of polymicrobial infections is still debated. The purpose of this study was to evaluate the effect of virulent enterococci in the presence or absence of Escherichia coli strains in the in vivo Caenorhabditis elegans model.
Principal Findings
This study demonstrated that there was a synergistic effect on virulence when an association of enterococci and E. coli (LT50 = 1.6 days±0.1 according to the tested strains and death of nematodes in 4 days±0.5) was tested in comparison with enterococci alone (LT50 = 4.6 days±0.1 and death in 10.4 days±0.6) or E. coli alone (LT50 = 2.1±0.9 and deaths 6.6±0.6) (p<0.001). In addition, there was a relation between the virulence of E. faecalis strains alone and the virulence potential of the association with E. coli strains. Finally, in the presence of avirulent E. coli strains, enterococci have no effect (LT50 = 4.3±0.5 and deaths in 10.8±0.8), independently of the level of their own virulence, demonstrating that the ‘enterococci effect’ only occurred in the presence of virulent E. coli strains.
Conclusion
This study allows a better understanding of a bacterial cooperation. Moreover, it could help to optimize the antibiotic regimen during polymicrobial infections.
doi:10.1371/journal.pone.0003370
PMCID: PMC2557124  PMID: 18843374
16.  Molecular Detection of Cellulosimicrobium cellulans as the Etiological Agent of a Chronic Tongue Ulcer in a Human Immunodeficiency Virus-Positive Patient 
Journal of Clinical Microbiology  2005;43(8):4269-4271.
Ulcerations appeared on the tongue of a 48-year-old human immunodeficiency virus-positive man. Histological findings of the biopsy specimen and the fact that the patient had resided in Louisiana led us to suspect “American histoplasmosis.” A new ulcer appeared while the patient was being treated with itraconazole, and the gene for 16S rRNA of Cellulosimicrobium cellulans was amplified. The lesions healed during treatment with oral penicillin and azithromycin.
doi:10.1128/JCM.43.8.4269-4271.2005
PMCID: PMC1234009  PMID: 16081997
17.  Emergence and Spread of Three Clonally Related Virulent Isolates of CTX-M-15-Producing Escherichia coli with Variable Resistance to Aminoglycosides and Tetracycline in a French Geriatric Hospital 
Antimicrobial Agents and Chemotherapy  2004;48(10):3736-3742.
Three types of multidrug-resistant Escherichia coli isolates, called GEN S, GEN R, and AMG S, according to their three different aminoglycoside resistance patterns, were responsible for urinary tract colonization or infection in 87, 12, and 13 new patients, respectively, in a French 650-bed geriatric hospital over a 13-month period. The three E. coli types belonged to the same clone and phylogenetic group (group B2) and had identical transferable plasmid contents (a 120-kb plasmid), β-lactam and fluoroquinolone resistance genotypes (blaTEM-1B, blaCTX-M-15, and double mutations in both the gyrA and the parC genes), and virulence factor genotypes (aer, fyuA, and irp2). They disseminated in the geriatric hospital, where the antibiotics prescribed most often were fluoroquinolones and ceftriaxone, but not in the affiliated acute-care hospital, where isolation precautions were applied to the transferred patients. Thus, E. coli isolates, both CTX-M-type β-lactamase producers and fluoroquinolone-resistant isolates, might present a new challenge for French health care settings.
doi:10.1128/AAC.48.10.3736-3742.2004
PMCID: PMC521882  PMID: 15388428
18.  Genetic and Biochemical Characterization of the Chromosomal Class A β-Lactamases of Raoultella (formerly Klebsiella) planticola and Raoultella ornithinolytica 
Enterobacterial strains of Raoultella spp. display a penicillinase-related β-lactam resistance pattern suggesting the presence of a chromosomal bla gene. From whole-cell DNA of Raoultella planticola strain ATCC 33531T and Raoultella ornithinolytica strain ATCC 31898T, bla genes were cloned and expressed into Escherichia coli. Each gene encoded an Ambler class A β-lactamase, named PLA-1 and ORN-1 for R. planticola and R. ornithinolytica, respectively. These β-lactamases (291 amino acids), with the same pI value of 7.8, had a shared amino acid identity of 94%, 37 to 47% identity with the majority of the chromosome-encoded class A β-lactamases previously described for Enterobacteriaceae, and 66 to 69% identity with the two β-lactamases LEN-1 and SHV-1 from Klebsiella pneumoniae. However, the highest identity percentage (69 to 71%) was found with the plasmid-mediated β-lactamase TEM-1. PLA-1, which displayed very strong hydrolytic activity against penicillins, also displayed significant hydrolytic activity against cefepime and, to a lesser extent, against cefotaxime and aztreonam, but there was no hydrolytic activity against ceftazidime. Such a substrate profile suggests that the Raoultella β-lactamases PLA-1 and ORN-1 should be classified into the group 2be of the β-lactamase classification of K. Bush, G. A. Jacoby, and A. A. Medeiros (Antimicrob. Agents Chemother. 39:1211-1233, 1995). The highly homologous regions upstream of the blaPLA-1A and blaORN-1A genes comprised a nucleotide sequence identical to the −35 region and another one very close to the −10 region of the blaLEN-1 gene. From now on, as the bla gene sequences of the most frequent Raoultella and Klebsiella species are available, the bla gene amplification method can be used to differentiate these species from each other, which the biochemical tests currently carried out in the clinical laboratory are unable to do.
doi:10.1128/AAC.48.1.305-312.2004
PMCID: PMC310189  PMID: 14693555
19.  New Klebsiella oxytoca β-Lactamase Genes blaOXY-3 and blaOXY-4 and a Third Genetic Group of K. oxytoca Based on blaOXY-3 
The two genetic groups (oxy-1 and oxy-2) previously identified in the Klebsiella oxytoca taxon are recognizable by four independent molecular markers: (i) ERIC-1R profiles, (ii) 16S ribosomal DNA (rDNA) signature sequences, (iii) singular nucleotides in a defined fragment of the rpoB gene, and (iv) the type of the strain's blaOXY gene (i.e., blaOXY-1 or blaOXY-2). K. oxytoca strains SG266 and SG271 could not be classified into these genetic groups based on their ERIC-1R profile and blaOXY gene sequence. With regard to the gene identity percentages between the blaOXY-1 and blaOXY-2 gene groups (86.8% ± 0.4%) and within a blaOXY gene group (>99%), it was concluded that the blaOXY gene of strain SG271 was representative of a new blaOXY gene group (blaOXY-3), since the mean identity percentages between it and the two blaOXY gene groups were 85.5% ± 0.2% and 84.4% ± 0.4%, respectively. Since the corresponding percentages were 95.0% ± 0.4% and 86.2% ± 0.3% for strain SG266, it was impossible to classify its blaOXY gene, which was therefore named blaOXY-4. The 16S rDNA signature sequences of the two strains could be determined only after cloning experiments. The SG266 clones displayed the same signature sequence as that of the genetic group oxy-1, whereas the SG271 clones displayed three different 16S rDNA signature sequences that also differed from those of the two genetic groups. Singular nucleotides were found within the rpoB sequence of the two strains, allowing for their distinction from the two genetic groups. All of these results, combined with those previously obtained by the ERIC-1R PCR method, indicate that strain SG271 is representative of a new K. oxytoca genetic group (oxy-3), whereas strain SG266 could not be classified.
doi:10.1128/AAC.47.9.2922-2928.2003
PMCID: PMC182611  PMID: 12936995
20.  Enterobacterial Repetitive Intergenic Consensus 1R PCR Assay for Detection of Raoultella sp. Isolates among Strains Identified as Klebsiella oxytoca in the Clinical Laboratory 
Journal of Clinical Microbiology  2003;41(4):1740-1742.
The enterobacterial repetitive intergenic consensus 1R PCR method, which provided recognizable profiles for reference strains of the three species of Raoultella and the two genetic groups of Klebsiella oxytoca, was applied to 19 clinical isolates identified as K. oxytoca. By this method, as confirmed by species-specific gene sequencing, two Raoultella ornithinolytica and two unclassifiable K. oxytoca isolates were identified.
doi:10.1128/JCM.41.4.1740-1742.2003
PMCID: PMC153902  PMID: 12682174
21.  Promoters P3, Pa/Pb, P4, and P5 Upstream from blaTEM Genes and Their Relationship to β-Lactam Resistance 
Antimicrobial Agents and Chemotherapy  2002;46(12):4035-4037.
Using an isogenic system, we have determined the impact that the four promoters known to control blaTEM gene expression have on β-lactamase activity. For both TEM-1 and TEM-30, this activity gradually increased in relation to the presence of promoters P3, Pa/Pb, and P4 upstream of the corresponding gene. Promoter P5, only found upstream of the blaTEM-1B gene, was related to the highest expression of this gene.
doi:10.1128/AAC.46.12.4035-4037.2002
PMCID: PMC132779  PMID: 12435720
24.  Emergence and Spread in French Hospitals of Methicillin-Resistant Staphylococcus aureus with Increasing Susceptibility to Gentamicin and Other Antibiotics 
Journal of Clinical Microbiology  1999;37(11):3452-3457.
Oxacillin (methicillin) resistance in methicillin-resistant Staphylococcus aureus (MRSA) is associated with an increased incidence of resistance to other antibiotics, which has increased since it was first reported in 1969. In 1992 a new phenotype of MRSA arose in France; this was characterized by a heterogeneous expression of resistance to oxacillin and susceptibility to various antibiotics, including gentamicin but also tetracycline, minocycline, lincomycin, pristinamycin, co-trimoxazole, rifampin, and fusidic acid. In French hospitals a longitudinal nationwide surveillance of antibiotic resistance in S. aureus has allowed for the detection of changes in antibiotic susceptibility profiles. Seven French clinical laboratories (six from the mainland and one from the West Indies) reported the results of susceptibility testing of 57,347 S. aureus strains isolated in their institutes between 1992 and 1998. Over a 7-year period the incidence of isolation of gentamicin-susceptible MRSA (GS-MRSA) strains has steadily increased to represent, in 1998, 46.8 to 94.4% of the MRSA strains, irrespective of the overall incidence of MRSA. Two predominant types recognized by pulsed-field gel electrophoresis (PFGE) accounted for the majority of the GS-MRSA in different mainland hospitals, both differing from the predominant type observed in the French West Indies. Some GS-MRSA and gentamicin-resistant MRSA (GR-MRSA) strains had closely related PFGE profiles, and hybridization studies confirmed the lack in GS-MRSA of the aac6′-aph2" gene, which confers resistance to all aminoglycosides, with conservation of the ant4′ gene, which confers resistance to kanamycin, tobramycin, and amikacin. Thus, it is likely that certain GS-MRSA strains could have emerged from GR-MRSA strains by excision or deletion of the aac6′-aph2" gene.
PMCID: PMC85665  PMID: 10523533
25.  Discriminatory Detection of Inhibitor-Resistant β-Lactamases in Escherichia coli by Single-Strand Conformation Polymorphism-PCR 
Plasmid-mediated mechanisms, comprising TEM hyperproduction, TEM derivative production, and OXA production, lead to amoxicillin-clavulanic acid resistance in enterobacteria. The ability of the single-strand conformation polymorphism (SSCP)-PCR method to differentiate the genes encoding inhibitor-resistant β-lactamases was evaluated with three blaTEM primer pairs. The blaTEM genes, which were known to be different on the basis of their nucleotide sequences (blaTEM-1A, blaTEM-1B, blaTEM-2, blaTEM-30, blaTEM-32, and blaTEM-35), were identified as different by their electrophoretic mobilities. The blaTEM-33, blaTEM-34, blaTEM-36, blaTEM-37, blaTEM-38, and blaTEM-39 genes, whose sequence differences have been established by oligotyping, displayed different SSCP profiles for different fragments, suggesting genetic differences in addition to those defined by oligotyping. Confirmed by sequencing, these additional genetic events concerned silent mutations at certain positions and, notably, a G→T transversion at position 1 of the −10 consensus sequence in blaTEM-34, blaTEM-36, blaTEM-37, and blaTEM-39. Applied to eight clinical isolates of Escherichia coli resistant to amoxicillin-clavulanic acid, the SSCP method detected TEM-1 in three strains and TEM-30, TEM-32, and TEM-35 in three other strains, respectively. A novel TEM derivative (TEM-58) was detected in another strain, and the deduced amino acid sequence showed two substitutions: Arg244Ser, which is known to confer amoxicillin-clavulanic acid resistance in TEM-30, and Val261Ile, which has not been described previously. The eighth strain produced an OXA β-lactamase. Given the discriminatory power and the applicability of SSCP-PCR, this method can be proposed as a means of following the evolution of the frequencies of the different inhibitor-resistant β-lactamases.
PMCID: PMC105559  PMID: 9559800

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