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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.  Escherichia coli ST131, an Intriguing Clonal Group 
Clinical Microbiology Reviews  2014;27(3):543-574.
In 2008, a previously unknown Escherichia coli clonal group, sequence type 131 (ST131), was identified on three continents. Today, ST131 is the predominant E. coli lineage among extraintestinal pathogenic E. coli (ExPEC) isolates worldwide. Retrospective studies have suggested that it may originally have risen to prominence as early as 2003. Unlike other classical group B2 ExPEC isolates, ST131 isolates are commonly reported to produce extended-spectrum β-lactamases, such as CTX-M-15, and almost all are resistant to fluoroquinolones. Moreover, ST131 E. coli isolates are considered to be truly pathogenic, due to the spectrum of infections they cause in both community and hospital settings and the large number of virulence-associated genes they contain. ST131 isolates therefore seem to contradict the widely held view that high levels of antimicrobial resistance are necessarily associated with a fitness cost leading to a decrease in pathogenesis. Six years after the first description of E. coli ST131, this review outlines the principal traits of ST131 clonal group isolates, based on the growing body of published data, and highlights what is currently known and what we need to find out to provide public health authorities with better information to help combat ST131.
PMCID: PMC4135899  PMID: 24982321
4.  Genomic Definition of Hypervirulent and Multidrug-Resistant Klebsiella pneumoniae Clonal Groups 
Emerging Infectious Diseases  2014;20(11):1812-1820.
We created a Web-accessible genome database to enable rapid extraction of genotype, virulence, and resistance information from sequences.
Multidrug-resistant and highly virulent Klebsiella pneumoniae isolates are emerging, but the clonal groups (CGs) corresponding to these high-risk strains have remained imprecisely defined. We aimed to identify K. pneumoniae CGs on the basis of genome-wide sequence variation and to provide a simple bioinformatics tool to extract virulence and resistance gene data from genomic data. We sequenced 48 K. pneumoniae isolates, mostly of serotypes K1 and K2, and compared the genomes with 119 publicly available genomes. A total of 694 highly conserved genes were included in a core-genome multilocus sequence typing scheme, and cluster analysis of the data enabled precise definition of globally distributed hypervirulent and multidrug-resistant CGs. In addition, we created a freely accessible database, BIGSdb-Kp, to enable rapid extraction of medically and epidemiologically relevant information from genomic sequences of K. pneumoniae. Although drug-resistant and virulent K. pneumoniae populations were largely nonoverlapping, isolates with combined virulence and resistance features were detected.
PMCID: PMC4214299  PMID: 25341126
Klebsiella pneumoniae; hypervirulent; invasive infections; multidrug resistance; clonal groups; emerging clones; genome evolution; multilocus sequence typing; MLST; diagnostics; high-throughput sequencing; bacteria
5.  Four Main Virotypes among Extended-Spectrum-β-Lactamase-Producing Isolates of Escherichia coli O25b:H4-B2-ST131: Bacterial, Epidemiological, and Clinical Characteristics 
Journal of Clinical Microbiology  2013;51(10):3358-3367.
A total of 1,021 extended-spectrum-β-lactamase-producing Escherichia coli (ESBLEC) isolates obtained in 2006 during a Spanish national survey conducted in 44 hospitals were analyzed for the presence of the O25b:H4-B2-ST131 (sequence type 131) clonal group. Overall, 195 (19%) O25b-ST131 isolates were detected, with prevalence rates ranging from 0% to 52% per hospital. Molecular characterization of 130 representative O25b-ST131 isolates showed that 96 (74%) were positive for CTX-M-15, 15 (12%) for CTX-M-14, 9 (7%) for SHV-12, 6 (5%) for CTX-M-9, 5 (4%) for CTX-M-32, and 1 (0.7%) each for CTX-M-3 and the new ESBL enzyme CTX-M-103. The 130 O25b-ST131 isolates exhibited relatively high virulence scores (mean, 14.4 virulence genes). Although the virulence profiles of the O25b-ST131 isolates were fairly homogeneous, they could be classified into four main virotypes based on the presence or absence of four distinctive virulence genes: virotypes A (22%) (afa FM955459 positive, iroN negative, ibeA negative, sat positive or negative), B (31%) (afa FM955459 negative, iroN positive, ibeA negative, sat positive or negative), C (32%) (afa FM955459 negative, iroN negative, ibeA negative, sat positive), and D (13%) (afa FM955459 negative, iroN positive or negative, ibeA positive, sat positive or negative). The four virotypes were also identified in other countries, with virotype C being overrepresented internationally. Correspondingly, an analysis of XbaI macrorestriction profiles revealed four major clusters, which were largely virotype specific. Certain epidemiological and clinical features corresponded with the virotype. Statistically significant virotype-specific associations included, for virotype B, older age and a lower frequency of infection (versus colonization), for virotype C, a higher frequency of infection, and for virotype D, younger age and community-acquired infections. In isolates of the O25b:H4-B2-ST131 clonal group, these findings uniquely define four main virotypes, which are internationally distributed, correspond with pulsed-field gel electrophoresis (PFGE) profiles, and exhibit distinctive clinical-epidemiological associations.
PMCID: PMC3811668  PMID: 23926164
6.  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.
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
7.  Different Factors Associated with CTX-M-Producing ST131 and Non-ST131 Escherichia coli Clinical Isolates 
PLoS ONE  2013;8(9):e72191.
To determine factors associated with CTX-M-producing ST131 Escherichia coli which is the worldwide predominant lineage among CTX-M-producing E. coli isolates.
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.
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.
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.
PMCID: PMC3762868  PMID: 24023730
8.  Phylogenetic Distribution of CTX-M- and Non-Extended-Spectrum-β-Lactamase-Producing Escherichia coli Isolates: Group B2 Isolates, Except Clone ST131, Rarely Produce CTX-M Enzymes 
Journal of Clinical Microbiology  2012;50(9):2974-2981.
Escherichia coli is the species most frequently associated with clinical infections by extended-spectrum-β-lactamase (ESBL)-producing isolates, with the CTX-M ESBL enzymes being predominant and found in genetically diverse E. coli isolates. The main objective of this study was to compare, on the basis of a case-control design, the phylogenetic diversity of 152 CTX-M-producing and 152 non-ESBL-producing clinical E. coli isolates. Multilocus sequence typing revealed that even though CTX-M enzymes were largely disseminated across the diversity of E. coli isolates, phylogenetic group B2 showed a particularly heterogeneous situation. First, clone ST131 of group B2 was strongly associated with CTX-M production (55 [79%] of 70 isolates), with CTX-M-15 being predominant. Second, the remaining members of group B2 were significantly less frequently associated with CTX-M production (9 [12%] of 75) than E. coli phylogenetic groups A, B1, and D (88 [55%] of 159). CTX-M-producing ST131 E. coli isolates were significantly more frequent in patients hospitalized in geriatric wards or long-term care facilities. Besides, the non-ESBL ST131 isolates significantly more frequently showed resistance to penicillins than the non-ESBL, non-ST131 isolates did. In conclusion, the present study emphasizes the particular antimicrobial resistance and epidemiologic characteristics of clone ST131 within group B2, which could result from the higher antibiotic exposure of this clone, as it is the predominant clone of group B2 carried in the human gut.
PMCID: PMC3421780  PMID: 22760036
9.  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.
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
10.  Virulence Potential and Genomic Mapping of the Worldwide Clone Escherichia coli ST131 
PLoS ONE  2012;7(3):e34294.
Recently, the worldwide propagation of clonal CTX-M-15-producing Escherichia coli isolates, namely ST131 and O25b:H4, has been reported. Like the majority of extra-intestinal pathogenic E. coli isolates, the pandemic clone ST131 belongs to phylogenetic group B2, and has recently been shown to be highly virulent in a mouse model, even though it lacks several genes encoding key virulence factors (Pap, Cnf1 and HlyA). Using two animal models, Caenorhabditis elegans and zebrafish embryos, we assessed the virulence of three E. coli ST131 strains (2 CTX-M-15- producing urine and 1 non-ESBL-producing faecal isolate), comparing them with five non-ST131 B2 and a group A uropathogenic E. coli (UPEC). In C. elegans, the three ST131 strains showed intermediate virulence between the non virulent group A isolate and the virulent non-ST131 B2 strains. In zebrafish, the CTX-M-15-producing ST131 UPEC isolates were also less virulent than the non-ST131 B2 strains, suggesting that the production of CTX-M-15 is not correlated with enhanced virulence. Amongst the non-ST131 B2 group isolates, variation in pathogenic potential in zebrafish embryos was observed ranging from intermediate to highly virulent. Interestingly, the ST131 strains were equally persistent in surviving embryos as the non-ST131-group B2 strains, suggesting similar mechanisms may account for development of persistent infection. Optical maps of the genome of the ST131 strains were compared with those of 24 reference E. coli strains. Although small differences were seen within the ST131 strains, the tree built on the optical maps showed that these strains belonged to a specific cluster (86% similarity) with only 45% similarity with the other group B2 strains and 25% with strains of group A and D. Thus, the ST131 clone has a genetic composition that differs from other group B2 strains, and appears to be less virulent than previously suspected.
PMCID: PMC3311635  PMID: 22457832
11.  Patient's Origin and Lifestyle Associated with CTX-M-Producing Escherichia coli: A Case-Control-Control Study 
PLoS ONE  2012;7(1):e30498.
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.
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.
PMCID: PMC3267726  PMID: 22299043
12.  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
13.  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.
PMCID: PMC2944602  PMID: 20679507
14.  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.
PMCID: PMC2937738  PMID: 20592153
15.  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
16.  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.
PMCID: PMC2786640  PMID: 19828750
17.  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
18.  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.
PMCID: PMC2593250  PMID: 18842941
19.  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
20.  Virulent Synergistic Effect between Enterococcus faecalis and Escherichia coli Assayed by Using the Caenorhabditis elegans Model 
PLoS ONE  2008;3(10):e3370.
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.
This study allows a better understanding of a bacterial cooperation. Moreover, it could help to optimize the antibiotic regimen during polymicrobial infections.
PMCID: PMC2557124  PMID: 18843374
21.  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.
PMCID: PMC1234009  PMID: 16081997
22.  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.
PMCID: PMC521882  PMID: 15388428
23.  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.
PMCID: PMC310189  PMID: 14693555
24.  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.
PMCID: PMC182611  PMID: 12936995
25.  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.
PMCID: PMC153902  PMID: 12682174

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