[Enterobacteriaceae and beta-lactams : wild susceptibility patterns]. / Entérobactéries et bêta-lactamines : phénotypes de résistance naturelle.

Four susceptibility patterns of wild types of enterobacteria against old beta-lactams including aminopenicillins, carboxypenicillins and first-generation cephalosporins were individualized during the 1980s : susceptible, penicillinase low level, cephalosporinase and a combination of penicillinase and cephalosporinase. Such indirect detection of a mechanism of resistance allowed an interpretative reading for this class of antibiotics. At the present time, seven susceptibility patterns were proposed for this family of gram negative bacilli. Nevertheless, an analysis of results in terms of MICs and diameters of inhibition zone sizes of the main bacterial species of enterobacteria, mainly obtained from the databank of European Committee on Antimicrobial Susceptibility Testing (EUCAST), compared to that observed when overproducing strains were isolated in vivo and in vitro and to the type of beta-lactamase identified and their amino acid sequences conducted to a proposal of five susceptibility patterns. The fifth wild type individualized in several enterobacteria since 2005 is related to the synthesis of various chromosomal extended-spectrum beta-lactamases (ESBL) which hydrolyze many beta-lactams including oxyimino-cephalosporins such as ceftriaxone or cefotaxime. Their expression in a wild strain is characteristic and conducted to our interest for their role as progenitors of the transferable CTM-M types. Otherwise, a medical biologist must consider the possibility of selection of a mutant with a chromosomal overproduced beta-lactamase. But within the same beta-lactam susceptibility pattern such as for Klebsiella pneumoniae and K. oxytoca or Citrobacter amalonaticus, the spectrum of inactivation will be highly variable according to the type of enzyme overproduced. Finally, a nice synergy observed between clavulanic acid and cefotaxime or ceftriaxone or even aztreonam does not mean anytime a transferable ESBL. In some cases according to the result of enterobacterial identification, the epidemiological impact will be very low, because without multidrug resistance (MDR).

[Lactobacillus delbrueckii: probable agent of urinary tract infections in very old women]. / Lactobacillus delbrueckii : probable cause d'infections urinaires chez la femme très âgée.

Lactobacillus urinary tract infection (UTI) seems exceptionally reported. Nevertheless, with the introduction of a chromogenic medium UriSelect 4, eight cases of UTI in old women (mean of 81.2 years) mediated by Lactobacillus delbrueckii identified by DNA sequencing were reported between 2007 and 2009.

[Beta-lactamases of Gram negative bacteria: never-ending clockwork!]. / Beta-lactamases de bacilles à Gram négatif: le mouvement perpétuel!

The acquired resistance against the wide-spectrum and highly stable beta-lactams including third-generation cephalosporins (3GC) and carbapenems is constinuously increasing and widespead with the discovery of various plasmid-encoded, or genes cassette or integrons coding for a novel beta-lactamase, always a major mechanism of resistance. To explain resistance against 3GC, with the continuing story with TEM and SHV mutated enzymes, several types of ESBL (class A) emerge the CTX-M type, at least CTX-M-40, but also other non predominant types intitled BES, GES, PLA, PER, VEB. The wider resistance including 3GC, cephamycins and beta-lactamase inhibitor is correlated to synthesis of transferable cephalosporinases (class C) usually located in the chromosome but mobilized from Enterobacter spp., Citrobacter freundii, Hafnia alvei, Morganella morganii, Aeromonas caviae. Such genes encoded the following types: ACC-1, ACT-1, CFE-1, CMY group, DHA-1, FOX group, MIR-1, MOX-1. Finally the resistance against carbapemens e.g. imipenem originally restricted to Pseudomonas aeruginosa, then to Acinetobacter baumannii and finally to enterobacteria is related to production of novel enzymes (classes B, D and A) denominated IMP, VIM SME, GIM, OXA, KPC. A striking exemple of evolution towards more and more resistance is given by Salmonella, even from animal origins, a great threat fo public health. So far it appears necessary to perform molecular approaches to identify such enzymatic production. Finally because the absence of real new drugs, the discovery of some progenitors of the gene beta-lactamase, a strict control of beta-lactam antibiotics must be provide not only in medecine or veterinary field but also in agriculture, including aquaculture for example.

Effect of simulated patient death on emergency worker's anxiety: a cluster randomized trial.

OBJECTIVE: Simulation-based teaching offers promising and diverse teaching possibilities. We aim to assess whether the death of the manikin increased anxiety amongst learner compared to similar simulation-based course where the manikin stays alive. METHODS: We conducted a cluster randomized study amongst multidisciplinary teams of emergency workers. Teams of physicians, nurses, and healthcare assistants were randomly assigned to participate in a simulation-based course where the simulated patient died (death group) or not (life group). We assessed anxiety at 1 month after the teaching using Spielberger STAI-state anxiety questionnaire. We compared reduction of anxiety when facing a life-threatening situation in both groups. RESULTS: We included 25 teams for a total of 129 participants. We analysed 63 participants in the death group and 57 in the life group. Baseline characteristics were similar in both groups, including baseline anxiety (STAI-state score 39.6 (7.8) in the death group vs 38.6 (7.1) in the life group). We report a significant reduction in both groups 1 month after the training: 6.6 (7.8) vs 6 (8.0), mean difference 0.5 (-2.4; 3.4). At 3 months, we report a significant greater reduction of anxiety in the death group (mean difference 4 [0.1; 7.9]). CONCLUSION: We observed in our sample that unexpected simulated patient death did not increase anxiety amongst multidisciplinary emergency workers.

[Identification of the beta lactamase R-TEM of Pseudomonas aeruginosa]. / Identification de la beta lactamase R-TEM chez Pseudomonas aeruginosa

This paper is dealing with the enzymatic problem raised by two strains of Ps. aeruginosa resistant to classical beta lactam antibiotics including carbenicillin. These two strains hydrolyse all these antibiotics. In both cases, we have shown the simultaneous biosynthesis of two enzymes: an inducible and chromosome cephalosporinase frequently found in this germ, and a constitutive beta lactamase, with a penicillinase activity which has been identified with the extrachromosomic beta lactamase R-TEM. These two enzymes have been separated by affinity chromatography, characterized by their kinetic constants given by computerized microacidimetry, and their isoelectric points which are respectively 9.2 for the cephalosporinase and 5.40 for the penicillinase R-TEM. Isoelectric focussing also shows the separation of these two enzymes.

Detection, distribution and inhibition of Branhamella catarrhalis beta-lactamases.

Beta-lactamase-producing isolates of Branhamella catarrhalis were first detected in France in 1977. The frequency of beta-lactamase producers has increased, especially since 1980. An agar iodometric test, a fast chromogenic test and an acidimetric test were used to assess the beta-lactamase-producing capabilities of 188 isolates of B. catarrhalis obtained mainly from sputum and the pharynx. Data from the first 2 procedures indicated positive beta-lactamase activity for all 49 strains of B. catarrhalis identified, but there were some discrepancies in the acidimetric test results. Evidence from a diffusion technique showed significant increases in the inhibition diameters surrounding filter discs impregnated with amoxycillin in the presence of clavulanic acid, or with ampicillin in the presence of sulbactam, compared with discs of the penicillins used alone. Two types of enzyme activity emerged from examination of isoelectric focusing patterns. Type I, having pI values of 5.35, 5.55 and 5.85, accounted for 87.2% of the enzyme-producing isolates. Type II, with pIs of 5.5, 5.9 and 6.25, occurred in 12.8% of isolates and appeared to be less widely distributed. The beta-lactamase inhibitors clavulanic acid and sulbactam in combination with benzylpenicillin produced potentiated effects, as demonstrated by significant reductions in MIC (33- and 44-fold decreases, respectively). Higher concentrations of each inhibitor similarly affected the MICs of amoxycillin. A weak synergy occurred with cefoxitin, a beta-lactamase-resistant beta-lactam antibiotic, and the 2 beta-lactamase inhibitors. Because B. catarrhalis has been shown to be a beta-lactamase-producing pathogenic organism, the addition of enzyme inhibitors, such as clavulanic acid and sulbactam, to standard therapy may be beneficial.

The analysis of five carbenicillin-hydrolysing enzymes by electrophoretic methods.

Five carbenicillin-hydrolysing enzymes (carbenicillinases, or CARB), PSE-4 (CARB-1), PSE-1 (CARB-2), CARB-3, CARB-4 and CARB-5, and the beta-lactamase PSE-2 were compared by analysing their isoelectric points (pI), electrophoretic mobilities (mR) and titration curves (pH gradient electrophoresis). The pI determined by isoelectric focusing were 4.3 (CARB-4), 5.3 (PSE-4/CARB-1), 5.7 (PSE-1/CARB-2), 5.75 (CARB-3), 6.1 (PSE-2) and 6.35 (CARB-5). Their mR were estimated by zone electrophoresis as congruent to 26 for PSE-1 (CARB-2), CARB-3 and CARB-5, congruent to 30 for PSE-2, congruent to 33 for PSE-4 (CARB-1) and congruent to 61 for CARB-4. Titration curve analyses indicated that (1) PSE-4 (CARB-1), PSE-1 (CARB-2), CARB-3 and CARB-5 are closely related variants differing by a few amino acid substitutions; (2) the qualitative titration curve of CARB-4 is different from those of PSE-4 (CARB-1), PSE-1 (CARB-2), CARB-3 and CARB-5, although their patterns are somewhat similar; and (3) PSE-2 has no structural relationship with any of the other carbenicillin-hydrolysing enzymes or carbenicillinases (CARB) studied. Electrophoretic methods, and in particular titration curve determination combined with other physicochemical and enzymatic data, allowed a rapid comparison of the molecular structures of the beta-lactamases, and hence their classification.

Antibiotic susceptibility in aerobic gram-negative bacilli isolated in intensive care units in 39 French teaching hospitals (ICU study).

OBJECTIVE: Evaluation of the distribution and antibiotic susceptibility of the aerobic gram-negative bacilli (AGNB) isolated from patients in intensive care units (ICU study). DESIGN AND SETTING: Microbiological study carried out in 1991 in 39 teaching hospitals. A standardized method was used to determine the minimum inhibitory concentrations of 12 antibiotics against 3366 strains of AGNB (close to 100 strains per hospital) during a period of 3 months. RESULTS: The 2773 initial strains (i.e., the first AGNB isolate for a given species and a given patient) were mainly isolated from the respiratory tract (34.4%), urinary tract (23%), or blood (9.6%) and were mainly Pseudomonas aeruginosa (22.9%), Escherichia coli (22%), Acinetobacter (9.7%), and Klebsiella pneumoniae (8.3%). E. coli was prominent in urine and blood and P. aeruginosa in the respiratory tract. Overall, the rate of susceptibility of AGNB was 58 to 65% to piperacillin, cefotaxime, and gentamicin; 69 to 75% to aztreonam, tobramycin, and ciprofloxacin; 83% to ceftazidime; and 91% to imipenem. The overall rates of susceptibility were higher for the initial strains isolated from blood than for those from the urinary or respiratory tracts, mostly reflecting differences in species distribution. Susceptibility rates were lower for the 593 repeat strains (i.e., all the subsequent isolates for a given species and a given patient) than for the initial strains, mostly due to the higher proportion of resistant species (P. aeruginosa 45.9%) but also due to the difference in susceptibility rates for some species-antibiotic combinations. Concomitant resistance (i.e., resistance to several antibiotics due to independent mechanisms of resistance) was marked between beta-lactams and aminoglycosides or quinolones, particularly in P. aeruginosa and K. pneumoniae. CONCLUSIONS: Rates of resistance in AGNB as a whole and in particular species (P. aeruginosa, Klebsiella), as well as frequency of concomitant resistance found in the French ICU study, were higher than those found in ICU studies conducted with the same methodology in Belgium, The Netherlands, and Germany, which may reflect differences in case mix.

Construction by polymerase chain reaction and use of intragenic DNA probes for three main types of transferable beta-lactamases (TEM, SHV, CARB) [corrected].

Intragenic DNA probes were synthesized by polymerase chain reaction using fragments of the genes of three major types of beta-lactamases (TEM, SHV, CARB) as templates. The TEM probe hybridized with the genes encoding TEM-1, TEM-2 and six extended-spectrum related enzymes (TEM-3 to TEM-7, TEM-2O) in colony hybridizations and Southern-blot analysis. The SHV probe hybridized with the genes for SHV-1, OHIO-1 and four derived extended-spectrum beta-lactamases (SHV-2, SHV-3, SHV-4 and SHV-5). The CARB probe hybridized with the genes for PSE-1 (CARB-2), PSE-4 (CARB-1), CARB-3 and CARB-4. None of the probes hybridized with genes for any of eight oxacillin-hydrolysing enzymes, PSE-2, OXA-1 to OXA-7, ROB-1 and chromosomal beta-lactamases of various Enterobacteriaceae (except Klebsiella pneumoniae) and Pseudomonas aeruginosa. Investigations of Escherichia coli clinical isolates using these probes indicate the presence of a novel type of extended-spectrum, transferable beta-lactamase.

Substitution of alanine for aspartate at position 179 in the SHV-6 extended-spectrum beta-lactamase.

SHV-6 was previously identified by its susceptibility pattern and biochemical criteria in a clinical isolate of Klebsiella pneumoniae which was resistant to ceftazidime. It contains only a single point difference with the beta laSHV-1 gene as determined by PCR amplification and nucleotide sequencing. This is the result of a single amino acid substitution, Ala for Asp, at position 179. Directed mutagenesis experiments have shown this substitution to confer selective resistance to ceftazidime in the TEM family.

Biochemical, immunological and physicochemical comparisons between OHIO-1 and four SHV-type beta-lactamases.

The biochemical, immunological and physicochemical properties of the beta-lactamase OHIO-1 were compared to those of four beta-lactamases commonly found in Klebsiella pneumoniae: SHV-1, SHV-3 and the beta-lactamases of strains GN 11-03 and GN 422. The substrate profile of SHV-1, OHIO-1 and of the beta-lactamases GN 11-03 and GN 422 were similar, while that of SHV-3 appeared comparable to that of the extended spectrum SHV-2. Moreover, anti-TEM-1 serum inactivated OHIO-1 as well as SHV-1 and the beta-lactamases of strains GN 11-03 and GN 422. Analysis of the electrophoretic mobilities, isoelectric points and titration curves demonstrated that OHIO-1 and the 4 other beta-lactamases examined were closely related variants. From these findings it appears that OHIO-1 could be classified among the SHV-type beta-lactamases.

Cloning and sequencing of the gene encoding the AmpC beta-lactamase of Morganella morganii.

The chromosomal beta-lactamase gene of a clinical isolate of Morganella morganii was cloned in Escherichia coli and sequenced. The beta-lactamase had a pI of 7.4 and conferred a typical AmpC susceptibility pattern. The insert obtained was found to encode a protein of 379 amino acids. Its deduced amino acid sequence revealed it to be a class C beta-lactamase: 39-56% identity with chromosomal AmpC beta-lactamases of Serratia marcescens, Yersinia enterocolitica, Citrobacter freundii, Enterobacter cloacae and Escherichia coli; and 37-56% identity with plasmid-mediated beta-lactamases (MOX-1, CMY-1, FOX-1, ACT-1, LAT-1, BIL-1 and CMY-2). The ampC gene was linked to a gene only part of which (450 bp) was cloned homologous to the regulatory ampR genes of chromosomal class C beta-lactamases.

Incidence of two virulence factors (aerobactin and mucoid phenotype) among 190 clinical isolates of Klebsiella pneumoniae producing extended-spectrum beta-lactamase.

Because outbreaks of multiple-resistant Klebsiella pneumoniae isolates producing extended-spectrum beta-lactamases were recently observed in French hospitals, the presence of virulence factors was examined for (i) phenotype by bioassay for aerobactin production and by culture for the mucoid phenotype, and (ii) genotype using intragenic probes of respectively 2-kb BglII and 235-bp BamHI-BglII fragments and dot-blotting among 190 unreplicated K. pneumoniae clinical isolates issued from 25 French hospitals and producing different types of extended-spectrum beta-lactamases (TEM-related enzymes: TEM-3, TEM-4, CAZ-1, CAZ-2, TEM-8, or SHV-related enzymes: SHV-2, SHV-3, SHV-4). Only 3.7% and 7% of K. pneumoniae isolates produced aerobactin and mucoid phenotypes respectively, unrelated to type of beta-lactamase. Only 2% had both factors. No discordance was reported according to the detection method tested. The low prevalence of such virulence factors seems to indicate they were not involved in dissemination of nosocomial K. pneumoniae isolates producing an extended-spectrum beta-lactamase.

Klebsiella oxytoca: resistance to aztreonam by overproduction of the chromosomally encoded beta-lactamase.

Aztreonam-resistant Klebsiella oxytoca strain SL7811 was selected on agar containing 1 microgram of aztreonam per ml from a susceptible strain SL781. The MICs for the resistant mutant towards penicillins, aztreonam and ceftriaxone were much higher, to cefotaxime slightly higher and to ceftazidime unchanged. Synthesis of beta-lactamase was 223-fold greater in the mutant compared with the susceptible strain. SL781 and its resistant mutant SL7811 produced beta-lactamase with the same isoelectric point and substrate profile. The beta-lactamase genes from SL781 and SL7811 were cloned in plasmid pBGS18 giving pBOF-1 and pBOF-4 respectively. The sequences of the two putative promoters indicated two modifications in the resistant plasmid pBOF-4: a transversion (G-->T) in the first base of the -10 consensus sequence and a deletion of one C residue four base pairs upstream of the -10 hexamer.

Novel transferable extended-spectrum beta-lactamase (SHV-6) from Klebsiella pneumoniae conferring selective resistance to ceftazidime.

A clinical isolate of Klebsiella pneumoniae sensu lato isolated from throat and a blood culture taken from a neutropenic patient treated for 2 weeks with ceftazidime and vancomycin was resistant to ceftazidime (MIC: 32 micrograms/ml) and moderately susceptible to aztreonam (MIC: 4 micrograms/ml). The isolate contained a plasmid of 180 kb which, when transferred to Escherichia coli by conjugation, conferred resistance to ceftazidime and tetracycline. The transconjugant had decreased susceptibility to ceftazidime (128-fold) and aztreonam (8-fold). Clavulanic acid and sulbactam each inhibited the resistance and clavulanic acid showed a synergistic effect when associated with ceftazidime and aztreonam. An extended-spectrum beta-lactamase with an isoelectric point of 7.6 was detected in the clinical isolates from blood and its transconjugant. This beta-lactamase showed similar substrate and inhibition profiles to SHV-1. In particular it did not hydrolyse ceftazidime. Hybridization with an intragenic probe for SHV-3 indicates that this beta-lactamase is an SHV-type enzyme. We propose that this novel CAZ-type extended-spectrum beta-lactamase be named SHV-6.

Two novel transferable extended-spectrum beta-lactamases from Klebsiella pneumoniae in Tunisia.

Two novel beta-lactamases conferring multiresistance to antibiotics including oxyimino beta-lactams have been identified in two nosocomial K. pneumoniae strains isolated in Tunis in 1986 and 1988. Both enzymes were encoded by ca. 150-kilobase plasmids. Donor and transconjugant strains producing these enzymes exhibited highly similar pattern of resistance (CTX phenotype) to beta-lactams including penicillins and oxyimino beta-lactams e.g. cefotaxime, ceftriaxone, ceftazidime, and aztreonam. High and variable synergy (16 to 1066-fold) was obtained when combined to 0.1 microgram/ml of clavulanate (beta-lactamase inhibitor). The isoelectric points of these two enzymes were 5.4 and 6.4. These beta-lactamases differed from TEM types by hydrolysis for cefotaxime or ceftriaxone but were inhibited by clavulanate and cloxacillin. DNA hybridization studies suggested that that the genes of these enzymes may be derived from genes encoding TEM-type enzymes.

Two variants of transferrable extended-spectrum TEM-beta-lactamase successively isolated from a clinical Escherichia coli isolate.

In a leukaemic patient presenting a septicaemia treated with ceftazidime and amikacin, two clinical Escherichia coli isolates distinguished by their level of resistance to oxyimino-beta-lactams were isolated at an interval of 24 h. The isolates were identified by biotyping and esterase electrophoretic typing and the two host strains were shown to be identical. However, each of these strains exhibited a different transferrable extended-spectrum beta-lactamase. These enzymes had different pI values (5.25 and 5.58), but were both blaTEM-1 mutants. The enzyme with pI 5.25 was identical to TEM-101 (TEM-12) (serine 162 substitution). The enzyme with pI 5.58 showed an additional amino acid substitution (lysine residue instead of an arginine at position 237) and was denominated TEM-23. These data indicate that point-mutations can be successively cumulated in vivo by blaTEM mutants, leading to expression of beta-lactamases with increased hydrolysis rates.

Characterisation of CMY-4, an AmpC-type plasmid-mediated beta-lactamase in a Tunisian clinical isolate of Proteus mirabilis.

A strain of Proteus mirabilis resistant to beta-lactams, including cefoxitin, was isolated from the urine of a woman from Tunisia. Its antibiotic susceptibility pattern and that of the Escherichia coli transconjugant suggested the presence of an AmpC-type beta-lactamase. Two bands of beta-lactamase activity (pI 5.4 and 9.2) were detected by isoelectric focusing. The nucleotide sequence of the gene encoding the AmpC-type enzyme was determined. The deduced amino acid sequence was 98-99% identical to CMY-3 and to those of the plasmid-mediated AmpC-type beta-lactamases originated from Citrobacter freundii and 97% identical to the chromosome-encoded beta-lactamase of a Tunisian clinical isolate of C. freundii. This enzyme differs from CMY-2 by one substitution (Arg for Trp at position 221) and from CMY-3 by two substitutions (Glu for Gly at position 42 and Ser for Asn at position 363) and we propose the denomination CMY-4.

Virulence factors (aerobactin and mucoid phenotype) in Klebsiella pneumoniae and Escherichia coli blood culture isolates.

We examined the presence of two virulence factors in 241 blood isolates of Klebsiella pneumoniae from patients hospitalized during 1989 and 1990 in 7 French hospitals, and 125 blood isolates of Escherichia coli from one hospital. Aerobactin was scored phenotypically and genotypically with an intragenic DNA probe of 2 kb. The mucoid phenotype was assessed by culture on trypticase soy agar and by genotypic analysis (intragenic DNA probe of 235 bp). Only 6% K. pneumoniae isolates were aerobactin-positive with no significant variation according to geographical location while 20% of K. pneumoniae isolates displayed the mucoid phenotype, with a significant variation according to hospital. Aerobactin was always associated with the mucoid phenotype. The frequency of aerobactin production but not mucoid phenotype (14%) was higher among E. coli isolates (48%). They harbored two types of large plasmids. Intraperitoneal injection into mice of 10(3) cfu of K. pneumoniae producing both virulence factors demonstrated that capsular serotype K2 was the more virulent K23 and K28.