Jean Duval

TRANSFERABLE ENZYMATIC RESISTANCE TO THIRD-GENERATION CEPHALOSPORINS DURING NOSOCOMIAL OUTBREAK OF MULTIRESISTANT KLEBSIELLA PNEUMONIAE

Klebsiella pneumoniae strains that were resistant to third-generation cephalosporins and amikacin were recovered from 62 of 395 patients (15.7%) during 1986. 25 isolates (40%) caused urinary tract infections. The outbreak involved three intensive care units (54 isolates), and spread from one unit to another and then to four wards (8 isolates). K pneumoniae of various serotypes and strains of different Enterobacteriaceae demonstrating the same antibiotic resistance pattern were isolated, which suggests dissemination of an R-factor. The isolates had low-level resistance to third-generation cephalosporins (mode minimum inhibitory concentration of cefotaxime, 2 mg/l) but remained sensitive to cephamycins. Cefotaxime was effective in cases of uncomplicated urinary tract infection, but failed in major infections at other sites. 1-5 mg/l of the beta-lactamase inhibitors clavulanic acid or sulbactam restored normal activity to cefotaxime against the multiresistant strains. Resistance to third-generation cephalosporins was mediated by a new broad-spectrum enzyme of isoelectric point 6.3. Resistance to beta-lactams and aminoglycosides was transferable to Escherichia coli. The emergence of transferable enzymatic resistance to newer beta-lactams in K pneumoniae strains indicates a major risk of spread of such resistance to otherwise sensitive strains.

Extrahepatic Immunologic Manifestations in Chronic Hepatitis C and Hepatitis C Virus Serotypes

Extrahepatic immunologic abnormalities have been shown to occur frequently in patients with chronic hepatitis C virus (HCV) infection. Hepatitis C virus now appears to cause those cases of mixed cryoglobulinemia that were previously considered essential [1-5]. Indeed, HCV RNA has been detected in the serum specimens of about 90% of patients with essential mixed cryoglobulinemia [1, 2, 4]. In addition, cryoglobulin is found, usually at low levels, in the serum specimens of one third to one half of patients with chronic hepatitis C [6, 7]; rheumatoid factor, which may play a role in cryoglobulinemia, is present in the serum specimens of about 70% of patients [6]. Various autoantibodies have been seen in the serum of 40% to 50% of patients with chronic HCV infection [6, 8], and HCV has been associated with cases of autoimmune thyroiditis [9]. Salivary gland lesions, characterized by lymphocytic capillaritis, are seen in about half of patients and are sometimes associated with lymphocytic sialadenitis resembling that of the Sjogren syndrome [6]. Finally, HCV may cause the chronic liver disease frequently associated with lichen planus [10]. Recently, sequences of different HCV variants were classified into different genotypes on the basis of overall sequence similarity [11-22]. A consensus nomenclature for HCV genotypes has been proposed [23], in which the six HCV genotypes identified so far are numbered in the order of their discovery. Within each genotype, subtypes have been identified by lower case letters, which are also given in order of discovery [23]. Correspondence among the classifications reported so far is presented in Table 1. Different techniques for determining HCV genotype have been developed in recent months. Currently, in addition to sequencing the genome, investigators can use three techniques based on the polymerase chain reaction (PCR). The technique described by Okamoto and colleagues [24, 25] is based on a nested PCR amplification of the HCV genome and uses primers located in the core region: The first round of PCR uses a pair of universal (non-type-specific) primers and the second uses a pair of type-specific primers. The method of McOmish and colleagues [17, 18] is based on PCR amplification of the 5 noncoding region of the genome done with a pair of universal primers, followed by enzymatic digestion of the amplified products and analysis of their restriction fragment length polymorphism. Stuyver and colleagues described a line probe assay for the determination of HCV genotypes [22], in which a PCR amplification is done using universal primers located in the 5 noncoding region of the genome. This is followed by hybridization of the amplified products to oligonucleotide probes attached as parallel bands on nitrocellulose strips. On the other hand, a serotyping immunoenzymatic assay to detect genotype-specific antibodies directed to epitopes encoded by the NS4 region of the HCV genome has been developed [26]. This technique, in its present form, allows the differentiation of HCV serotypes 1, 2, and 3, which correspond to HCV genotypes 1, 2, and 3 in the consensus nomenclature [23]. Table 1. Correspondence between the Major Published Classification Systems for Hepatitis C Virus Genotypes* Several studies indicate that particular HCV genotypes are associated with more severe liver disease and poorer response to interferon- therapy [27-30]. The factors determining immunologic abnormalities in patients with chronic hepatitis C are largely unknown. We used a serotyping assay to study whether the occurrence of extrahepatic immunologic abnormalities in patients with chronic hepatitis C is serotype dependent. Methods Patients Fifty-nine consecutive patients with chronic hepatitis C were prospectively studied. Thirty-four were men and 25 were women; their mean age was 52 years (range, 18 to 77 years). In all cases, the diagnosis of chronic hepatitis C was based on long-term elevation of serum alanine aminotransferase levels in the blood, positive serologic markers of HCV infection (found using second-generation enzyme-linked immunosorbent assay and recombinant immunoblot assay, Ortho Diagnostic Systems, Raritan, New Jersey), and the absence of any other cause of chronic liver disease. Specimens obtained by percutaneous liver biopsy showed chronic active hepatitis in all 56 patients tested and associated cirrhosis in 15 of the 56 (27%). Before any treatment was given, serum specimens were tested for cryoglobulin, rheumatoid factor, and many antitissue antibodies, and biopsy of labial salivary glands was done. Hepatitis C virus serotype was determined in all patients by immunoenzymatic assay. Study Methods Detection of Cryoglobulinemia Venous blood (20 mL) was taken from fasting patients in a room at 37 C, allowed to clot at this temperature, and then separated by centrifugation. After centrifugation, the supernatant was removed from the serum, incubated at 4 C for 8 days, and examined daily for cryoprecipitation. Detection of Rheumatoid Factor Rheumatoid factor was measured using a nephelometer analyzer (BNA, Behring, Marburg, Germany); polystyrene particles coated with human globulin were agglutinated when mixed with samples containing rheumatoid factor. Normal values were those less than 18 IU/mL. Detection of Autoantibodies Antinuclear, anti-smooth muscle, type 1 anti-liver-kidney microsomal (anti-LKM1), and antimitochondrial antibodies were detected by indirect immunofluorescence using air-dried cryostat sections from rat or mouse livers and kidneys and HEp-2 cells (Kallestad, Chaska, Minnesota) as substrates. Antithyroid microsomal antibodies were detected by indirect immunofluorescence using surgical specimens of human thyrotoxic thyroid as substrate. In all cases, the classic Weller and Coon indirect immunofluorescence method was used with fluorescein-labeled goat immunoglobulin directed to IgG, IgA, and IgM (Pasteur Diagnostics, Marnes la Coquette, France) as a second layer [31]. The serum specimens were tested undiluted for anti-DNA antibodies, at a 1/5 dilution for antimicrosomal antibodies, and at a 1/10 dilution for other antibodies. The titers were established using increasing dilutions up to 1/2560. Antithyroglobulin antibodies were detected using an hemagglutination kit (Thymune-T, Wellcome Diagnostics, Dartford, United Kingdom). Labial Salivary Gland Examination All biopsies were done in macroscopically normal mucosa. The samples were fixed in Bouin fluid, embedded in paraffin, and stained with hematoxylin-eosin-safranin. All sections were examined blind by two pathologists and graded according to the Chisholm and Mason classification system [32]. Determination of Serotypes The 59 serum specimens in our study were tested for the presence of serotype-specific antibodies using the recently developed enzyme immunoassay [26]. A series of eight branched peptides, synthesized from two antigenic regions of HCV genotypes 1, 2, and 3, were used to coat polypropylene microtiter wells overnight at 4 C. After washing, the wells were blocked with 150 L of blocking solution (phosphate-buffered saline, 0.1% Tween 20, and 2% bovine serum albumin) for 1 hour at room temperature. Blocking assays were done using mixes of type-specific peptides at a final concentration of 1 mg/mL (for example, 100:1 excess over that used to coat the wells). Plasma specimens from the 59 patients were diluted in the blocking solution and 100 L were added to antigen-coated and blocked wells. The first incubation was done overnight at 4 C. Plates were washed four times in phosphate-buffered saline and 0.1% Tween 20 and then incubated with horseradish peroxidase-conjugated anti-human IgG (1/20 000 in phosphate-buffered saline and 0.1% Tween 20 for 1 hour at room temperature). The plates were finally washed four times in phosphate-buffered saline and 0.1% Tween 20 and incubated with substrate (50 g of O-phenylenediamine per milliliter and 0.1% H2O2 [30 volumes] for 30 minutes in the dark at room temperature). Optical densities were read at 490 nm; values ranged from 100 to 2000 mU. Results Prevalence of Immunologic Abnormalities Our results are presented in Table 2. Cryoglobulin was found in the serum specimens of 20 of the 56 patients tested (36%), and rheumatoid factor was present at abnormal levels in 42 of the 59 patients (71%). Table 2. Prevalence of the Different Immunologic Abnormalities according to Hepatitis C Virus Serotype At least one type of antitissue antibody was detected in the serum specimens of 24 of the 59 patients (41%). Thirteen patients (22%) had serum antinuclear antibodies and 13 (22%) had anti-smooth muscle antibodies at a significant titer (greater than 1/40). Anti-LKM1 antibodies were found in 3 patients (5%) and antithyroid antibodies were found in 5 (8%); 4 of these 5 had antithyroglobulin and 1 had antithyroid microsomes. No antimitochondrial antibodies were found. Labial salivary gland biopsies were done in those 49 of the 59 patients who had no contraindication and who gave informed consent; lesions were found in 24 of them (49%). In all patients, these lesions were characterized by lymphocytic capillaritis, as previously described [6]. In 7 patients (14%), they were associated with more severe lesions, grades 3 and 4 by the Chisholm and Mason classification (lymphocytic sialadenitis) [32], and resembled the lymphocytic sialadenitis of the Sjogren syndrome (14%). Only one of the patients with salivary gland lesions had a mild case of the ocular sicca syndrome shown by the Schirmer test. The prevalences of the different immunologic abnormalities did not vary significantly according to the presence of cirrhotic findings in liver specimens. Hepatitis C Virus Serotypes Thirty-five of the 59 patients (59%) were infected with HCV serotype 1, 6 (10%) were infected with serotype 2, and 7 (12%) were infected with serotype 3 (serotypes are here described using the proposed consensus nomenclature [23]). Two

Transferable vancomycin and teicoplanin resistance in Enterococcus faecium.

Enterococcus faecium BM4165 and BM4178, isolated from immunocompromised patients, one treated with vancomycin, were inducibly resistant to high levels of the glycopeptide antibiotics vancomycin and teicoplanin but susceptible to the new lipopeptide daptomycin (LY146032). Strain BM4165 was also resistant to macrolidelincosamide-streptogramin B-type (MLS) antibiotics. The genes conferring resistance to glycopeptides and to MLS antibiotics in strain BM4165 were carried on plasmids pIP819 and pIP821, respectively; pIP819 also carried genes that encoded resistance to MLS antibiotics. The two plasmids, which were distinct although related, were self-transferable to other E. faecium strains. Plasmid pIP819 could also conjugate to E. faecalis, Streptococcus sanguis, S. pyogenes, S. lactis, and Listeria monocytogenes, in which it conferred inducible glycopeptide resistance, but not to S. aureus. Glycopeptide-inactivating activity was not detected, and the biochemical mechanism of resistance remains unknown. Based on this first report of transferable resistance to glycopeptides, we anticipate dissemination of resistance to these antibiotics in gram-positive cocci and bacilli in which it can be phenotypically expressed. Images

Vancomycin resistance gene vanC is specific to Enterococcus gallinarum.

Nearly all strains of Enterococcus gallinarum are resistant to low levels of vancomycin. The glycopeptide resistance gene vanC from E. gallinarum BM4174 has recently been cloned and sequenced. A probe specific for vanC hybridized with a 2.7-kb EcoRI and a 4.5-kb HindIII fragment of total DNA from the 42 strains of E. gallinarum studied. No homology was detected with DNA of strains belonging to other species intrinsically resistant to vancomycin, including Enterococcus casseliflavus, a species that expresses a vancomycin resistance phenotype similar to that of E. gallinarum. No hybridization with DNA of enterococcal strains with acquired resistance to high or low levels of vancomycin was observed. The specificity of the vanC probe allowed us to distinguish E. gallinarum from 12 other species of enterococci, indicating that this probe is a useful tool for species identification within the genus Enterococcus. Images

Identification of the satA gene encoding a streptogramin A acetyltransferase in Enterococcus faecium BM4145.

Enterococcus faecium BM4145, a clinical isolate from urine, was resistant to streptogramin group A antibiotics by inactivation. The strain harbored a plasmid containing a gene, satA, responsible for this resistance; this gene was cloned and sequenced. It encoded SatA, a protein deduced to be 23,634 Da in mass and homologous with a new family of chloramphenicol acetyltransferases described in Agrobacterium tumefaciens, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. The similarity of SatA to other acetyltransferases, LacA (thiogalactoside acetyltransferase) and CysE (serine acetyltransferase) from E. coli, and to two putative acetyltransferases, NodL from Rhizobium leguminosarum and Urf1 from E. coli, was also observed in a region considered to be the enzymes active site. Acetylation experiments indicated that acetyl coenzyme A was necessary for SatA activity and that a single acetylated derivative of pristinamycin IIA was produced. Other members of the streptogramin A group such as virginiamycin M and RP54476 were also substrates for the enzyme. We conclude that resistance to the streptogramin A group of antibiotics in E. faecium BM4145 is due to acetylation by an enzyme related to the novel chloramphenicol acetyltransferase family. Images

Hepatitis C virus infection and autoimmune thrombocytopenic purpura

BACKGROUND/AIMS Chronic hepatitis C virus infections are often associated with extra-hepatic immunological manifestations, including various autoimmune disorders. The aims of this study were: (i) to determine the prevalence of hepatitis C virus markers in patients with autoimmune thrombocytopenic purpura, and (ii) to determine whether a relationship could exist between autoimmune thrombocytopenic purpura and hepatitis C virus infections. METHODS One hundred and thirty-nine patients with autoimmune thrombocytopenic purpura (45 men, 94 women, mean age 42 years, range 16-90) were studied. RESULTS Anti-HCV antibodies were sought in their first and last available cryopreserved sera. In case of seropositivity, all their available cryopreserved sera were tested for anti-HCV antibodies and for HCV-RNA. Anti-HCV antibodies were detected in 14 of the 139 patients (10%). Four patients had transient anti-HCV seropositivity due to passive transfer of anti-HCV antibodies secondary to the infusion of intravenous immunoglobulin concentrates. Three patients seroconverted during follow up, due to intravenous drug use in one case, transfusion of non-HCV-screened blood units in one case, and infusion of intravenous immunoglobulins in one case. Seven patients had chronic hepatitis C discovered at the same time as autoimmune thrombocytopenic purpura. In two of them, hepatitis C virus transmission was the consequence of autoimmune thrombocytopenic purpura treatment but, in five cases, hepatitis C virus infection predated autoimmune thrombocytopenic purpura, so that the role of hepatitis C virus in autoimmune thrombocytopenic purpura could be suggested. CONCLUSIONS Whereas hepatitis C virus does not appear to be the main etiological agent of autoimmune thrombocytopenic purpura can be envisaged. On the other hand, treatment of autoimmune thrombocytopenic purpura or autoimmune thrombocytopenic purpura-related symptoms by blood product infusion can be complicated by hepatitis C virus transmission.

Antimicrobial susceptibility of Pediococcus spp. and genetic basis of macrolide resistance in Pediococcus acidilactici HM3020.

We determined the MICs of 28 antimicrobial agents against 36 clinical strains of Pediococcus spp. (25 P. acidilactici, 9 P. pentosaceus, and 2 P. urinaeequi strains). Penicillin G, imipenem, gentamicin, netilmicin, erythromycin, clindamycin, rifampin, chloramphenicol, daptomycin, and ramoplanin were the most active. All strains of P. acidilactici were susceptible to novobiocin, whereas all isolates of P. pentosaceus were resistant. Novobiocin could therefore be helpful for differentiation of these two closely related species. P. acidilactici HM3020 was inducibly resistant to macrolide, lincosamide, and streptogramin B-type (MLS) antibiotics. Resistance was due to a determinant homologous to ermAM and carried by a nontransferable 46-kb plasmid, pVM20. This plasmid was structurally distinct from two enterococcal MLS resistance plasmids, pIP819 and pAM beta 1. The 34 strains of P. acidilactici and P. pentosaceus were resistant to tetracycline, and total DNA of these strains did not hybridize to probes specific for tetK, tetL, tetM, and tetO.

Plasmid-mediated resistance to lincomycin by inactivation in Staphylococcus haemolyticus.

Staphylococcus haemolyticus BM4610 was resistant to high levels of lincomycin and susceptible to macrolides, clindamycin, and streptogramins. This resistance phenotype, not previously reported for a human clinical isolate, was due to inactivation of the antibiotic. The gene conferring resistance to lincomycin in strain BM4610 was carried by a 2.5-kilobase plasmid, pIP855, which was cloned in Escherichia coli. Plasmid pIP855 caused inactivation of both lincomycin and clindamycin in S. haemolyticus and in E. coli but conferred detectable resistance to lincomycin only in S. haemolyticus and to clindamycin only in E. coli. Images

In vitro [3H]-erythromycin binding to Staphylococcus aureus

Characteristics of erythromycin binding to Staphylococcus aureus were determined by using kinetics and equilibrium binding experiments. Both methods yielded identical values of the dissociation constant, i.e. 0.1 muM. This value was in accord with that found with a bacterial extract of ribosomes which are the organelles where erythromycin exerts its action. This good agreement shows that the dissociation constant of erythromycin determined with intact bacteria is a good reflect of specific bacterial receptors of macrolides, i.e. ribosomes. In addition, mechanism of uptake of the antibiotic by Staphylococcus aureus was investigated. Passive diffusion process was shown to be mainly responsible for this phenomenon.

In vivo selection during pefloxacin therapy of a mutant of Staphylococcus aureus with two mechanisms of fluoroquinolone resistance.

Staphylococcus aureus BM4626 (ciprofloxacin MIC, 0.5 microgram/ml) and BM4627 (ciprofloxacin MIC, 32 microgram/ml) were isolated from the same patient before and during pefloxcin therapy for septic tibial nonunion, respectively. The two strains had similar serotypes and indistinguishable phage types and SmaI-generated restriction fragment length polymorphisms. Portions of the gyrA (codons 60 to 120) and the gyrB (codons 420 to 480) genes of each clinical isolate were amplified by PCR and sequenced. Strain BM4627 had a serine-to-leucine substitution resulting from a cytosine-to-thymidine mutation at codon 84 of gyrA relative to the sequence of the gyrA gene of BM4626. Norfloxacin accumulation, measured in a whole-cell uptake assay, was significantly lower in BM4627 than BM4626. These data indicate that double mutants can be selected in vivo under fluoroquinolone therapy.