A penicillin- and metronidazole-resistant Clostridium botulinum strain responsible for an infant botulism case.

The clinical course of a case of infant botulism was characterized by several relapses despite therapy with amoxicillin and metronidazole. Botulism was confirmed by identification of botulinum toxin and Clostridium botulinum in stools. A C. botulinum A2 strain resistant to penicillins and with heterogeneous resistance to metronidazole was isolated from stool samples up to 110 days after onset. Antibiotic susceptibility was tested by disc agar diffusion and MICs were determined by Etest. Whole genome sequencing allowed detection of a gene cluster composed of blaCBP for a novel penicillinase, blaI for a regulator, and blaR1 for a membrane-bound penicillin receptor in the chromosome of the C. botulinum isolate. The purified recombinant penicillinase was assayed. Resistance to ß-lactams was in agreement with the kinetic parameters of the enzyme. In addition, the ß-lactamase gene cluster was found in three C. botulinum genomes in databanks and in two of 62 genomes of our collection, all the strains belonging to group I C. botulinum. This is the first report of a C. botulinum isolate resistant to penicillins. This stresses the importance of antibiotic susceptibility testing for adequate therapy of botulism.

Why is antibiotic resistance a deadly emerging disease?

Evolution of bacteria towards resistance to antimicrobial agents, including multidrug resistance, is unavoidable because it represents a particular aspect of the general evolution of bacteria that is unstoppable. Therefore, the only means of dealing with this situation is to delay the emergence and subsequent dissemination of resistant bacteria or resistance genes. In this review, we will consider the biochemical mechanisms and the genetics that bacteria use to offset antibiotic selective pressure. The data provided are mainly, if not exclusively, taken from the work carried out in the laboratory, although there are numerous other examples in the literature.

Structural insights into the loss of penicillinase and the gain of ceftazidimase activities by OXA-145 ß-lactamase in Pseudomonas aeruginosa.

OBJECTIVES: We previously described extended-spectrum oxacillinase OXA-145 from Pseudomonas aeruginosa, which differs from narrow-spectrum OXA-35 by loss of Leu-155. The deletion results in loss of benzylpenicillin hydrolysis and acquisition of activity against ceftazidime. We report the crystal structure of OXA-145 and provide the basis of its switch in substrate specificity. METHODS: OXA-145 variants were generated by site-directed mutagenesis and purified to homogeneity. The crystal structure of OXA-145 was determined and molecular dynamics simulations were performed. Kinetic parameters were investigated in the absence and in the presence of sodium hydrogen carbonate (NaHCO3) for representative substrates. RESULTS: The structure of OXA-145 was obtained at a resolution of 2.3 Å and its superposition with that of OXA-10 showed that Trp-154 was shifted by 1.8 Å away from the catalytic Lys-70, which was not N-carboxylated. Addition of NaHCO3 significantly increased the catalytic efficiency against penicillins, but not against ceftazidime. The active-site cavity of OXA-145 was larger than that of OXA-10, which may favour the accommodation of large molecules such as ceftazidime. Molecular dynamics simulations of OXA-145 in complex with ceftazidime revealed two highly coordinated water molecules on the α- or ß-face of the acyl ester bond, between Ser-67 and ceftazidime, which could be involved in the catalytic process. CONCLUSIONS: Deletion of Leu-155 resulted in inefficient positioning of Trp-154, leading to a non-carboxylated Lys-70 and thus to loss of hydrolysis of the penicillins. Ceftazidime hydrolysis could be attributed to enlargement of the active site and to a catalytic mechanism independent of the carboxylated Lys-70.

Antimicrobial resistance of Listeria monocytogenes strains isolated from humans in France.

Susceptibility to antibiotics of 4,816 clinical L. monocytogenes strains isolated since 1926 was studied, and the temporal evolution of susceptibility to antibiotics was analyzed through several decades. The mechanisms of resistance in each resistant strain were studied. The prevalence of resistant strains was estimated at 1.27% among isolates from humans. Resistance to tetracyclines+ and fluoroquinolones was more common and has recently emerged. Although acquired resistance in clinical L. monocytogenes did not implicate clinically relevant antibiotics, the possibility of resistance gene transfers, the description of the first clinical isolate with high-level resistance to trimethoprim, and the recent increase in penicillin MICs up to 2 microg/ml reinforce the need for microbiological surveillance.

New combinations of mutations in VanD-Type vancomycin-resistant Enterococcus faecium, Enterococcus faecalis, and Enterococcus avium strains.

We studied the clinical isolates Enterococcus faecium NEF1, resistant to high levels of vancomycin (MIC, 512 microg/ml) and teicoplanin (MIC, 64 microg/ml); Enterococcus faecium BM4653 and BM4656 and Enterococcus avium BM4655, resistant to moderate levels of vancomycin (MIC, 32 microg/ml) and to low levels of teicoplanin (MIC, 4 microg/ml); and Enterococcus faecalis BM4654, moderately resistant to vancomycin (MIC, 16 microg/ml) but susceptible to teicoplanin (MIC, 0.5 microg/ml). The strains were distinct, were constitutively resistant via the synthesis of peptidoglycan precursors ending in D-alanyl-D-lactate, and harbored a chromosomal vanD gene cluster that was not transferable. New mutations were found in conserved domains of VanS(D): at T(170)I near the phosphorylation site in NEF1, at V(67)A at the membrane surface in BM4653, at G(340)S in the G2 ATP-binding domain in BM4655, in the F domain in BM4656 (a 6-bp insertion), and in the G1 and G2 domains of BM4654 (three mutations). The mutations resulted in constitutivity, presumably through the loss of the phosphatase activity of the sensor. The chromosomal Ddl D-Ala:D-Ala ligase had an IS19 copy in NEF1, a mutation in the serine (S(185)F) or near the arginine (T(289)P) involved in D-Ala1 binding in BM4653 or BM4655, respectively, and a mutation next to the lysine (P(180)S) involved in D-Ala2 binding in BM4654, leading to the production of an impaired enzyme. In BM4653 vanY(D), a new insertion sequence, ISEfa9, belonging to the IS3 family, resulted in the absence of D,D-carboxypeptidase activity. Strain BM4656 had a functional D-Ala:D-Ala ligase, associated with high levels of both VanX(D) and VanY(D) activities, and is the first example of a VanD-type strain with a functional Ddl enzyme. Study of these five clinical isolates, displaying various assortments of mutations, confirms that all VanD-type strains isolated so far have undergone mutations in the vanS(D) or vanR(D) gene, leading to constitutive resistance, but that the Ddl host ligase is not always impaired. Based on sequence differences, the vanD gene clusters could be assigned to two subtypes: vanD-1 and vanD-4.

Can pharmacokinetic-pharmacodynamic parameters provide dosing regimens that are less vulnerable to resistance?

Dissemination of antibiotic resistance in bacteria is associated with prescription of the corresponding drugs. Various pharmacokinetic-pharmacodynamic parameters have been developed with the intention of reducing the spread of resistance. In this review, it is considered whether dosing regimens based on these parameters can delay this spread. The evolution of bacterial resistance to antibiotics involves two successive but distinct and independent mechanisms. The first occurs by mutation in the genome, including the host chromosome and mobile accessory genetic elements such as plasmids or transposons, or, following acquisition of a resistance determinant from another bacterium, by horizontal gene transfer. These two genetic events happen by chance, which means that they do not rely on the presence of an antibiotic in the environment; that is, they are not induced, but simply revealed and propagated by the drugs. The second step is dissemination of resistance which can be due to the spread of bacteria (clonal epidemics), of replicons (plasmid epidemics) or of resistance determinants (gene epidemics). Resistance dissemination by each one of these three levels which superimpose in nature, is not only infectious but also exponential, since all three are associated with DNA replication (duplication) of the host chromosome, of a plasmid, or of a transposon. As opposed to emergence, dissemination is clearly associated with the selective pressure exerted by antibiotic prescription [1,2]. The consequence of this dual evolutionary pathway is that proper use of antibiotics will, at best, delay the spread of resistance. In this review, the pharmacokinetic-pharmacodynamic (PK-PD) parameters that are intended to lower resistance dissemination are considered exclusively.

[Qnr-type quinolone resistance in extended-spectrum beta-lactamase producing enterobacteria in Abidjan, Ivory Coast]. / Résistance aux quinolones de type qnr chez les entérobactéries productrices de bêta-lactamases à spectre élargi à Abidjan en Côte d'Ivoire.

The aim of the study was to show the emergence of the qnr genes in extended spectrum beta-lactamases producing enterobacteria in Abidjan between 2005 and 2006. The whole of 151 strains of extended spectrum beta-lactamases producing enterobacteria were studied: 64 Escherichia coli, 66 Klebsiella pneumoniae, seven Klebsiella oxytoca and 14 Enterobacter spp. isolated from various biological products and from in- and out-patients. The techniques of disks diffusion, double-disk synergy, E-test were respectively used for the antimicrobial susceptibility test, the detection of extended spectrum beta-lactamases and the minimal inhibiting concentration. The bla genes(SHV, TEM, CTXM groups 1, 2, 8, 9), and AmpC were determined by PCR and characterized by sequencing. A global prevalence of 27,2 % (41/151) and rates of 9,9, 14,6, 2,7 % for the qnr genes A, B, A and S were observed. The distribution was 42,9 % for Enterobacter spp, 31,2 % for Escherichia coli, 20,5 % for Klebsiella; 30 strains expressed at least two bla genes; four strains were associated with AmpC. The strains were resistant to the cotrimoxazole (97,6 %), to the céfépime (73,2 %), to the céfoxitine (56,1 %), to the imipénème (0 %) and 43,9 % to all the aminosides. This high qnr gene prevalence associated with several types of bla genes in epidemic matter, the high level of resistance to antibiotics make fear a high risk of the transmission of multi-resistants bacteria and challenge the authorities for a resistance monitoring policy.

Real-time PCR detection of gyrA and parC mutations in Streptococcus pneumoniae.

Fluoroquinolone resistance in Streptococcus pneumoniae mainly involves stepwise mutations predominantly in the parC and gyrA genes. We have developed a single-run real-time PCR assay for detection of the four most common mutations in the quinolone resistance-determining regions of these genes. This assay provides a useful tool for both clinical and epidemiological use.

Predictable and unpredictable evolution of antibiotic resistance.

Evolution of bacteria towards antibiotic resistance is unavoidable as it represents a particular aspect of the general evolution of bacteria. Thus, at the very best, the only hope we can have in the field of resistance is to delay dissemination of resistant bacteria or resistance genes. Resistance to antibiotics in bacteria can result from mutations in resident structural or regulatory genes or from horizontal acquisition of foreign genetic information. In this review, we will consider the predictable future of the relationship between bacteria and antibiotics.

Bactofection of lung epithelial cells in vitro and in vivo using a genetically modified Escherichia coli.

Bacteria-mediated gene transfer ('bactofection') has emerged as an alternative approach for genetic vaccination and gene therapy. Here, we assessed bactofection of airway epithelial cells in vitro and in vivo using an attenuated Escherichia coli genetically engineered to invade non-phagocytic cells. Invasive E. coli expressing green fluorescent protein (GFP) under the control of a prokaryotic promoter was efficiently taken up into the cytoplasm of cystic fibrosis tracheal epithelial (CFTE29o-) cells and led to dose-related reporter gene expression. In vivo experiments showed that following nasal instillation the vast majority of GFP-positive bacteria pooled in the alveoli. Further, bactofection was assessed in vivo. Mice receiving 5 x 10(8) E. coli carrying pCIKLux, in which luciferase (lux) expression is under control of the eukaryotic cytomegalovirus (CMV) promoter, showed a significant increase (P<0.01) in lux activity in lung homogenates compared to untransfected mice. Surprisingly, similar level of lux activity was observed for the non-invasive control strain indicating that the eukaryotic CMV promoter might be active in E. coli. Insertion of prokaryotic transcription termination sequences into pCIKLux significantly reduced prokaryotic expression from the CMV promoter allowing bactofection to be detected in vitro and in vivo. However, bacteria-mediated gene transfer leads to a significantly lower lux expression than cationic lipid GL67-mediated gene transfer. In conclusion, although proof-of-principle for lung bactofection has been demonstrated, levels were low and further modification to the bacterial vector, vector administration and the plasmids will be required.

Evaluation of non-automated techniques for phenotypic detection of VanA-type Staphylococcus aureus.

The study presented here was performed to evaluate the ability of various nonautomated in vitro susceptibility testing methods to detect glycopeptide resistance in the three presently identified strains of VanA-type methicillin-resistant Staphylococcus aureus. To this end, MICs were determined according to the guidelines of the National Committee for Clinical Laboratory Standards, the Comité de l'Antibiogramme de la Société Française de Microbiologie and the British Society for Antimicrobial Chemotherapy and by using the E-test, agar disc diffusion, agar screening plates, and ATB galleries. In each of these methods, vancomycin was more efficient than teicoplanin for detecting glycopeptide resistance.

Worldwide disseminated armA aminoglycoside resistance methylase gene is borne by composite transposon Tn1548.

The armA (aminoglycoside resistance methylase) gene, which confers resistance to 4,6-disubstituted deoxystreptamines and fortimicin, was initially found in Klebsiella pneumoniae BM4536 on IncL/M plasmid pIP1204 of ca. 90 kb which also encodes the extended-spectrum beta-lactamase CTX-M-3. Thirty-four enterobacteria from various countries that were likely to produce a CTX-M enzyme since they were more resistant to cefotaxime than to ceftazidime were studied. The armA gene was detected in 12 clinical isolates of Citrobacter freundii, Enterobacter cloacae, Escherichia coli, K. pneumoniae, Salmonella enterica, and Shigella flexneri, in which it was always associated with bla(CTX-M-3) on an IncL/M plasmid. Conjugation, analysis of DNA sequences, PCR mapping, and plasmid conduction experiments indicated that the armA gene was part of composite transposon Tn1548 together with genes ant3"9, sul1, and dfrXII, which are responsible for resistance to streptomycin-spectinomycin, sulfonamides, and trimethoprim, respectively. The 16.6-kb genetic element was flanked by two copies of IS6 and migrated by replicative transposition. This observation accounts for the presence of armA on self-transferable plasmids of various incompatibility groups and its worldwide dissemination. It thus appears that posttranscriptional modification of 16S rRNA confers high-level resistance to all the clinically available aminoglycosides except streptomycin in gram-negative human and animal pathogens.

Bacterial transfer of large functional genomic DNA into human cells.

Efficient transfer of chromosome-based vectors into mammalian cells is difficult, mostly due to their large size. Using a genetically engineered invasive Escherichia coli vector, alpha satellite DNA cloned in P1-based artificial chromosome was stably delivered into the HT1080 cell line and efficiently generated human artificial chromosomes de novo. Similarly, a large genomic cystic fibrosis transmembrane conductance regulator (CFTR) construct of 160 kb containing a portion of the CFTR gene was stably propagated in the bacterial vector and transferred into HT1080 cells where it was transcribed, and correctly spliced, indicating transfer of an intact and functional locus of at least 80 kb. These results demonstrate that bacteria allow the cloning, propagation and transfer of large intact and functional genomic DNA fragments and their subsequent direct delivery into cells for functional analysis. Such an approach opens new perspectives for gene therapy.

Clonal spread of pediatric isolates of ciprofloxacin-resistant, emm type 6 Streptococcus pyogenes.

Twenty-four community isolates of Streptococcus pyogenes resistant to ciprofloxacin and susceptible to levofloxacin, gatifloxacin, and moxifloxacin were studied. Sequence determination of the quinolone resistance-determining regions in the gyrA and parC genes revealed a T/G mutation in parC leading to a Ser79Ala substitution in ParC. All isolates were of the emm type 6, and 18 and 2 of them were indistinguishable or closely related, respectively, on the basis of pulsed-field gel electrophoresis.

In vitro antibacterial activity of moxifloxacin against hospital isolates: a multicentre study.

OBJECTIVE: To evaluate the in vitro antibacterial activity of moxifloxacinin in comparison to that of other fluoroquinolones (ciprofloxacin, ofloxacin and trovafloxacin). METHODS: A total of 2,196 strains was collected in 11 French hospitals in 1998. Minimum inhibitory concentrations (MICs) (mg/L) were determined by agar dilution and agar diffusion was performed with 5-microg discs. Internal quality control was carried out with genetically defined strains. RESULTS: MIC50s and MIC90s of moxifloxacin against nalidixic acid (NAL)-susceptible Enterobacteriaceae (n = 663) were 0.12 and 0.5. As for other quinolones, the activity of moxifloxacin (4-32) was reduced against NAL-intermediate and NAL-resistant strains (n = 222). MIC50s and MIC90s of moxifloxacin were 2 and 4 for ciprofloxacin-susceptible P. aeruginosa (n = 128); moxifloxacin had no activity against ciprofloxacin-resistant strains (n = 56). The activity of moxifloxacin was maintained against NAL-susceptible A. baumannii (n = 11; 0.032-0.125), but reduced against NAL-resistant strains (n = 30; 16-32). H. influenzae (n = 97) and M. catarrhalis (n = 40) were inhibited by low concentrations (0.03-0.06 and 0.06-0.25, respectively). Moxifloxacin had better activity (0.06-0.12) than other tested quinolones against methicillin-susceptible S. aureus strains (n = 110); ciprofloxacin-resistant strains (n = 85) (2-8) were usually methicillin-resistant. Moxifloxacin was moderately active against enterococci (n = 149) (E. faecalis: 0.5-16; E. faecium: 2-4). Streptococci (n = 194) and pneumococci (n = 136), including 70 penicillin G-intermediate or G-resistant strains, were inhibited by low concentrations (0.25-0.5 for each species). Based on the regression curve, tentative zone diameter breakpoints could be > or =21 and <18 mm for MIC breakpoints of < or =1 and >2 mg/L, respectively. CONCLUSIONS: While retaining activity against Enterobacteriaceae, moxifloxacin was moderately active against P. aeruginosa. Its activity was inferior to that of ciprofloxacin for these species. This study confirmed the comparatively high in vitro activity of moxifloxacin against Gram-positive cocci and other pathogens isolated from community-acquired respiratory tract infections.

Multiple antibiotic resistance gene transfer from animal to human enterococci in the digestive tract of gnotobiotic mice.

It has been proposed that food animals represent the source of glycopeptide resistance genes present in enterococci from humans. We demonstrated the transfer of vanA and of other resistance genes from porcine to human Enterococcus faecium at high frequency in the digestive tract of gnotobiotic mice. Tylosin in the drinking water favored colonization by transconjugants.

Frequency of isolation and antimicrobial susceptibility of bacterial pathogens isolated from patients with bloodstream infections: a French prospective national survey.

All bloodstream strains, total 1463, isolated during a 1 month period in 105 hospitals representing all geographical areas in France were collected to study their antimicrobial susceptibility. The three major species were Escherichia coli, Staphylococcus aureus and coagulase-negative staphylococci. Among the 242 S. aureus, 87 were resistant to methicillin and among those 99% were resistant to ciprofloxacin, 11.5% to gentamicin, 1% to quinupristin/dalfopristin and 8% were heterogeneously resistant to vancomycin. Study of the methicillin-resistant S. aureus indicated that 12 clones had disseminated in French hospitals, six being heterogeneously resistant to vancomycin. Among the Streptococcus pneumoniae, 43% showed decreased susceptibility to the penicillins and 42% to erythromycin. One isolate was highly resistant to fluoroquinolones. Gentamicin, cefotaxime, ciprofloxacin and gatifloxacin resistance was rare in Enterobacteriaceae with 95% of strains susceptible. The incidence of extended-spectrum beta-lactamases was quite low. Moreover more than 25% of Pseudomonas aeruginosa strains were resistant to ciprofloxacin and gentamicin. The magnitude of antibiotic resistance in bloodstream isolates, in particular Gram-positive bacteria, emphasizes the importance of hospital control measures, rational prescribing policies and new vaccine strategies.

Bacterial resistance to penicillin G by decreased affinity of penicillin-binding proteins: a mathematical model.

Streptococcus pneumoniae and Neisseria meningitidis have very similar mechanisms of resistance to penicillin G. Although penicillin resistance is now common in S. pneumoniae, it is still rare in N. meningitidis. Using a mathematical model, we studied determinants of this difference and attempted to anticipate trends in meningococcal resistance to penicillin G. The model predicted that pneumococcal resistance in a population similar to that of France might emerge after 20 years of widespread use of beta-lactam antibiotics; this period may vary from 10 to 30 years. The distribution of resistance levels became bimodal with time, a pattern that has been observed worldwide. The model suggests that simple differences in the natural history of colonization, interhuman contact, and exposure to beta-lactam antibiotics explain major differences in the epidemiology of resistance of S. pneumoniae and N. meningitidis.

Comparability of antimicrobial susceptibility test results from 22 European countries and Israel: an external quality assurance exercise of the European Antimicrobial Resistance Surveillance System (EARSS) in collaboration with the United Kingdom National External Quality Assurance Scheme (UK NEQAS).

The goal of this exercise was to organize external quality assurance (QA) of antibiotic susceptibility testing for laboratories participating in EARSS and to assess the comparability of susceptibility test results across countries, and guidelines. In September 2000, UK NEQAS distributed a set of three Streptococcus pneumoniae strains, two Staphylococcus aureus strains and one Streptococcus haemolyticus strain. Laboratories reported the guideline followed, the interpretation of the susceptibility test result and the MIC, if tested. In this study we considered results 'concordant' if the reported interpretation of the participating laboratory agreed with the designated interpretation of reference laboratories. Overall, 433 (92%) of 471 laboratories from 23 countries reported back. Of the 8685 tests that were assessed, 8322 (96%) were interpreted correctly by the participants. Concordance for detection of penicillin non-susceptibility in the three S. pneumoniae strains was 96%, 90% and 87%, respectively. Laboratories performed extremely well in detecting oxacillin resistance in the homogeneously methicillin-resistant S. aureus (MRSA) strain, but the concordance rate dropped from 100% to 77% in the heterogeneously resistant MRSA strain. Concordance for detection of teicoplanin resistance in the S. haemolyticus strain was 82%. We stratified concordance rates first for country and then for guideline used, but observed only minor differences among countries and guidelines. Quantitative methods yielding an MIC were more concordant than non-MIC methods for penicillin resistance in the S. pneumoniae strains (94% versus 79%). The NCCLS guideline was the most frequently followed, by 61% of laboratories from 19 countries. This exercise shows that, overall, countries participating in EARSS are capable of delivering susceptibility data of good quality. The comparability of susceptibility data for penicillin resistance in S. pneumoniae and for homogeneous methicillin resistance in S. aureus is satisfactory among European countries and across guidelines. However, we emphasize the importance of determining an MIC for suspected penicillin non-susceptible S. pneumoniae and for suspected glycopeptide non-susceptible S. aureus. Laboratories, particularly in some countries, may need to improve their capability to detect oxacillin resistance in heterogeneously resistant MRSA. For continuous external quality assessment we recommend that laboratories participate in national and international schemes with frequent distribution of control strains.