Carriage of Staphylococcus aureus and of gram-negative bacilli resistant to third-generation cephalosporins in cirrhotic patients: a prospective assessment of hospital-acquired infections.

OBJECTIVE: To study the relation between Staphylococcus aureus nasal and stool colonization, stool carriage of gram-negative bacilli resistant to third-generation cephalosporins (CephR), and subsequent infections during hospitalization. DESIGN: Prospective study. PATIENTS: 551 cirrhotic patients with 589 consecutive hospital stays. All patients were screened within 48 hours of admission; 589 nasal swabs, 417 stool specimens, and 589 urine samples were analyzed. RESULTS: Carriage rates were 18.8% for methicillin-sensitive S aureus (MSSA), 16.3% for methicillin-resistant S aureus (MRSA), and 13.7% for CephR. We observed 87 episodes of spontaneous bacterial peritonitis, 63 cases of bacteremia, and 167 urinary tract infections occurred. Only 1 case of bacteremia and 4 urinary tract infections due to CephR occurred in patients carrying the same organism in their stools. The risk of MRSA ascitic fluid infections, bacteremia, and urinary tract infections was 3.1% versus 1% (not significant), 8.3% versus 0.8% (P<.001), and 11.4% versus 0.6% (P<.001) in carriers and noncarriers, respectively. Pulsed-field gel electrophoresis (PFGE) of isolates from 16 patients infected by MSSA (3 cases) and MRSA (13 cases) demonstrated that the colonizing strains matched the invasive strains in the 3 MSSA cases and in 8 of 13 MRSA cases. CONCLUSION: Carriage of CephR strains is not associated with subsequent infection by these organisms in hospitalized cirrhotic patients. In contrast, MRSA carriage was an important risk factor for MRSA bacteremia and urinary tract infection.

A 5-year epidemiological study of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae isolates in a medium- and long-stay neurological unit.

Thirty-eight different strains of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae (ESBL Kp), isolated from urine and pus samples of 38 patients hospitalized in a medium- and long-stay neurology department between 1 January 1992 and 31 December 1996, were analysed by antibiotic resistance phenotyping, DNA macrorestriction by pulsed-field electrophoresis and isoelectric focusing of beta-lactamases. An epidemiological survey was conducted to identify risk factors for infection by ESBL Kp in this setting. The 38 isolates were distributed into 13 antibiotypes, three of which predominated (13, six and six isolates). The DNA macrorestriction pattern identified 15 genotypes, four of which predominated (11, six, four and four isolates). A combination of the two typing methods revealed several epidemic clones that emerged consecutively. Two main types of ESBL (SHV-2 and CTX-1) were identified by isoelectric focusing, the former predominating. The case-control study showed that the length of hospital stay, degree of malnutrition and dependency, and urinary sphincter status were the main factors significantly associated with ESBL Kp isolation.

Changes in nature and antibiotic resistance of bacteria causing peritonitis in cirrhotic patients over a 20 year period.

AIM: To assess all clinically and bacteriologically documented episodes of spontaneous bacterial peritonitis diagnosed in a single unit over a 20 year period, to identify changes in the nature and antibiotic resistance of the causative bacteria. SETTING: A specialist liver disease unit in a tertiary care centre. MATERIAL: Cultured ascitic fluid obtained in the course of 240 consecutive episodes of clinically and bacteriologically proven spontaneous bacterial peritonitis. Patient recruitment remained stable during the 20 year period in terms of the number of cirrhotic patients admitted and the severity of their condition. RESULTS: 78.7% of isolates were Enterobacteriaceae (Escherichia coli in 51%) and 19% were Gram positive cocci. Until 1979 all the Enterobacteriaceae had the wild phenotype, compared with only 50% at the end of the study period. Since 1993, 22% of Enterobacteriaceae have been resistant to third generation cephalosporins. Methicillin resistant staphylococci were only isolated after 1989. CONCLUSIONS: Changes in the epidemiology and antibiotic resistance of bacteria causing spontaneous bacterial peritonitis must be monitored for optimal treatment.

Epidemiology of severe hospital-acquired infections in patients with liver cirrhosis: effect of long-term administration of norfloxacin.

We performed a 5-year retrospective study to evaluate the effect of long-term administration of norfloxacin on the epidemiology of severe hospital-acquired infections in patients with advanced cirrhosis. Sixty-seven episodes of spontaneous bacterial peritonitis and 60 episodes of bacteremia occurred in, respectively, 46 patients (group 1a) and 52 patients (group 1b) who did not receive norfloxacin, while 23 and 17 episodes occurred in 21 patients (group 2a) and 17 patients (group 2b) during or within 10 days after long-term administration of norfloxacin. Enterobacteriaceae were more prevalent in groups 1a and 1b than in the other two groups (P < .001 and P < .01, respectively); conversely, staphylococci were more prevalent in groups 2a and 2b (P < .001 and P < .05, respectively). The rate of staphylococcal resistance to methicillin was 53.6% in groups 1a and 1b and 77.3% in groups 2a and 2b. We conclude that long-term norfloxacin administration to cirrhotic patients reduces the risk of gram-negative infections but increases the risk of severe hospital-acquired staphylococcal infections and of high-level resistance to antibiotics.

Enzyme electrophoresis combined with serogrouping for improved differentiation of Clostridium difficile strains.

We used enzyme electrophoresis to study a set of epidemiologically related and unrelated isolates of Clostridium difficile. The 53 strains belonged to the most frequent serogroups (A1, C, G, H and K). Nine electrophoretic profiles were defined on the basis of five enzymes, and two were characteristic of a single strain. Each serogroup was resolved into two or three different enzyme patterns. By combining the two methods we were able to resolve the strains into 12 types. There was an excellent correlation between enzyme electrophoresis and serogrouping data. This method may be of use in investigating nosocomial transmission.

Methicillin-resistant Staphylococcus aureus subtyping: interest of combined antibiotyping and esterase electrophoretic typing.

Ninety-four methicillin-resistant Staphylococcus aureus isolates (MRSA) were characterized by means of two typing methods, antibiotyping and esterase electrophoretic typing. Antibiotyping, recorded on the basis of susceptibility testing of 13 antimicrobial agents, allowed the description of 18 antibiotypes, four of which comprised 30, 14, 14 and 12 strains respectively. Esterase electrophoretic typing, based on esterase activity against seven synthetic substrates after polyacrylamide-agarose gel electrophoresis, led to the description of 12 electrophoretic types, two of which were predominant with 60 and 20 strains respectively. The combined use of both typing methods yielded 32 combinations, three of which were predominant with 21, 12 and 11 strains respectively. A good differentiation of strains was achieved, particularly when the antibiotype was correlated to the electrophoretic type. Thus, the combination of antibiotyping with esterase electrophoretic typing may be proposed as a well-suited method for the characterization of MRSA strains.

[Outbreak of infections of Klebsiella pneumoniae with extended spectrum beta-lactamase in a hospital unit with a long and medium stay]. / Epidémie d'infections à Klebsiella pneumoniae à beta-lactamase à spectre étendu dans un service de long et moyen séjour.

Fourteen extended-spectrum beta-lactamase (ES beta la) Klebsiella pneumoniae strains were isolated from 14 inpatients between February 1993 and February 1994, in a medium- and long-stay neurological unit. For this reason an epidemiological study was begun, based on strain typing and examination of patient files. Strain typing was carried out by two methods (i) the analysis of antibiotic resistance, showing 7 different antibiotypes among the 14 strains studied, (ii) the analysis of esterase and dehydrogenase electrophoretic polymorphism in polyacrylamide-agarose gel. The method was checked by analysing 11 Klebsiella pneumoniae strains with wild phenotype for beta-lactam antibiotics, which were isolated during the same period in the same unit. Simultaneously 6 other strains isolated during the same period in some other units of the hospital were analysed. Nine electrophoretic types were found among the 31 strains (wild and ES beta las). The analysis of the results showed that 8 isolates of the group of 14 ES beta las had the same antibiotype and electrophoretic type. This demonstrates that one epidemic strain was responsible for two outbreaks, the first one in April and the second one in August-September. A case control investigation was carried out to define the risk factors of infection. Files were examined for the 14 infected inpatients and for 20 control inpatients from the same unit during the same period. Statistical analysis was performed with Epi Info software 5 (CDC Atlanta). Length of stay, dependence and malnutrition levels, and urinary sphincter disfunction were the most significant risk factors.

Multilocus enzyme typing of human and animal strains of Clostridium perfringens.

Multilocus enzyme electrophoresis was developed to evaluate the genetic diversity of 71 human strains and 17 animal strains of Clostridium perfringens. Crude protein extracts, obtained by sonication of washed bacteria, were analyzed by polyacrylamide-agarose gel electrophoresis to characterize electrophoretic mobility variants of seven enzymes (esterase, glutamate dehydrogenase, glutamic-oxaloacetic transaminase, nucleoside phosphorylase, phosphoglucose isomerase, phosphoglucomutase, threonine dehydrogenase). Genetic diversity of the enzyme loci ranged from 0.340 to 0.813. Sixty-nine electrophoretic types were described among the 88 strains tested and the index of discrimination was 0.994. All strains were typable, and epidemiological relationships between isolates could be established. This method showed a fair correlation with esterase electrophoretic typing based on hydrolytic and electrophoretic polymorphism of esterases. This work demonstrates that multilocus enzyme polymorphism is a reliable and discriminant marker of genetic diversity of strains of C. perfringens.

Rapid emergence of quinolone resistance in cirrhotic patients treated with norfloxacin to prevent spontaneous bacterial peritonitis.

We carried out quantitative culturing of stools from 31 hospitalized alcoholic patients with cirrhosis and ascites, before treatment with 400 mg of norfloxacin per day, weekly for the first month, and then every 2 weeks thereafter for 15 to 229 days (median, 54 days). Members of the family Enterobacteriaceae virtually disappeared from the stools (< 10(2)/g), but treatment had little effect on enterococci. No selection of resistant organisms occurred in 15 patients, but the remaining 16 patients developed fecal organisms resistant to fluoroquinolones between days 14 and 43 of treatment (median, 25 days). Staphylococcus aureus was isolated four times, coagulase-negative Staphylococcus spp. were isolated six times, Citrobacter freundii was isolated four times, Enterobacter cloacae was isolated three times, Klebsiella oxytoca was isolated twice, Proteus rettgeri was isolated once, and untypeable streptococci were isolated six times. Some isolates persisted, while others were transient (one to seven consecutively positive cultures). The MICs of four quinolones (nalidixic acid, norfloxacin, ofloxacin, and ciprofloxacin) were determined by use of experimental microwell strips (ATB CMI; Biomerieux S.A.). All the strains isolated before treatment were susceptible to the four quinolones, with low MICs, whereas those isolated during norfloxacin treatment were highly resistant. Long-term norfloxacin administration thus carries a risk of disturbing the bacterial ecology in these patients, suggesting that digestive decontamination should no longer be prescribed routinely to cirrhotic patients with ascites.

Esterase electrophoretic polymorphism of human and animal strains of Clostridium perfringens.

Esterase electrophoretic polymorphism in human and animal strains of Clostridium perfringens was studied by using polyacrylamide-agarose gel electrophoresis. Five types of esterases, designated E-I to E-V and defined by their hydrolytic specificities toward five synthetic substrates, were found in protein extracts of bacteria grown without glucose (glucose-containing media allowed only the expression of esterase E-I). Mobility variants of esterase E-I, which hydrolyzes alpha- and beta-naphthyl acetates and butyrates, were used as a basis for the distribution of strains into 11 zymogroups. When all five types of esterases and their electrophoretic variants were considered, 77 electrophoretic types (ETs) could be described for the 89 strains tested. Animal strains did not constitute a distinctive subpopulation, as revealed by their distribution in the zymogroups and by clustering analysis. Statistical analysis also emphasized the importance of esterase E-IV (which hydrolyzes only naphthyl acetates) and esterase E-V (which hydrolyzes only alpha-naphthyl acetate) in clustering by the relatedness of the ETs. ETs allowed the epidemiological characterization of stool isolates recovered from elderly inpatient residents and from adolescent chronic-care psychiatric patients. These results indicate that esterase electrophoretic typing may be a marker for epidemiological and ecological analyses.

Use of electrophoretic polymorphisms of esterases for differentiation of Clostridium argentinense strains.

Esterase electrophoresis was used to study 10 strains of Clostridium argentinense, including 7 toxigenic and 3 nontoxigenic strains. On the basis of the electrophoretic mobilities and hydrolytic specificities toward five synthetic substrates, different esterase profiles could be defined for almost all strains, revealing the heterogeneity of bacterial clones. Therefore, electrophoretic polymorphism of esterases can be used for differentiation of C. argentinense in population genetic or epidemiological studies.

l-Methionine, a Precursor of Trace Methane in Some Proteolytic Clostridia.

The in vivo formation of methane and of several S-methyl volatile compounds from the terminal S-methyl group of l-methionine is reported for growing cultures of four Clostridium strains (C. hastiforme, C. histolyticum, C. subterminale, and Clostridium sp. strain DSM 1786). After growth in 5 ml of unamended medium, C. hastiforme formed the highest amount of methane (408 nmol per tube in the headspace). When the culture medium was amended with 100 mM l-[S-methyl-H(3)]methionine, the four strains formed [H(3)]methane (proportion in the methane peak, >85%) as well as methanethiol, dimethyl disulfide, dimethyl trisulfide, and S-methyl thioacetate labeled on the methyl moiety. Methanethiol is also a precursor of methane for Clostridium sp. strain DSM 1786. The trace methane formation observed for these four proteolytic, nonglucidolytic Clostridium strains can be of ecological interest, particularly in aquatic sediments and in the gastrointestinal tract of humans and animals. It can explain in part the trace methane formation which cannot be ascribed to methanogens sensu stricto.

Beta 2-microglobulin in liver cirrhosis: study of a local synthesis in ascitic fluid.

Beta 2-microglobulin determinations in ascitic fluid (A) and serum (S) collected on the same day, were performed in 24 patients suffering from alcoholic liver cirrhosis. Ascitic beta 2-m concentration varied from 0.4 to 4.6 mg/l for patients with a normal renal function. Much higher values were found in patients with chronic renal failure. No correlation could be established between ascitic beta 2-m level and the clinical evolution of the cirrhosis. Comparative measurements of beta 2-m S/A ratio and albumin, transferrin, total protein S/A ratios suggests a local synthesis of beta 2-m in ascitic fluid. This is confirmed by an immuno-cytochemical technique which reveals the localisation of beta 2-m in the cytoplasm of peritoneal cells. The presence of beta 2-m in ascitic fluid seems to be related to an ultrafiltration across the peritoneal membrane as well as a local polyclonal activation of the immune system.

Rapid diagnosis of gram negative urinary infections: identification and antimicrobial susceptibility testing in 24 hours.

A rapid method for diagnosing urinary tract infections, using identification and antimicrobial susceptibility testing that can be carried out in 24 hours, was devised. The method relies on direct inoculation of diluted urine (1/500) in the API 20 E and API ATB systems. Urine was simultaneously cultured on Columbia blood agar and on Drigalski agar to control the purity and for purposes of comparison. The results of this method and those obtained with a conventional method were compared by analysing 1352 urines. The results showed that all of the organisms were correctly identified using the conventional method, and susceptibility testing (rapid method) gave results that agreed with those of the classical method in 94% of cases, with major discrepancies in only 0.08% of cases. The rapid method applies only to monomicrobial infections.

Headspace gas chromatographic-mass spectrometric analysis of light hydrocarbons and volatile organosulphur compounds in reduced-pressure cultures of Clostridium.

A static headspace gas chromatographic method for the simultaneous separation of trace light hydrocarbons and volatile organosulphur compounds in gases of nineteen Clostridium cultures at reduced pressure is described. The separation was achieved on n-octane-Porasil C after sampling of the gaseous compounds in a PTFE loop without any pretreatment. Most peaks were identified by gas chromatography-mass spectrometry. The presence of methane and ethylene sulphide among Clostridium volatiles is confirmed and 3-methyl-1-butene, 2-methyl-2-butene, dimethyl trisulphide and S-methyl thioacetate are reported for the first time in the Clostridium group.

Gas chromatographic-mass spectrometric analysis of volatile amines produced by several strains of Clostridium.

A gas chromatographic--mass spectrometric technique is proposed for the analysis of volatile amines which were isolated from Clostridium cultures by vacuum distillation and concentrated as hydrochloride salts. Headspace sampling after alkalinization of the salts under vacuum was the most suitable for subsequent gas chromatographic analysis. With ammonia-loaded helium as carrier gas, methylamines were separated on 4.8% PEG 2OM + 0.3% potassium hydroxide on Carbopack B, and other volatile amines on 28% Pennwalt 223 + 4% potassium hydroxide on Gas-Chrom R. Bacterial volatile amines (dimethylamine, trimethylamine, isobutylamine, 3-methylbutylamine, etc.) were detected with a flame-ionization detector and identified by gas chromatography--mass spectrometry in electron-impact and chemical ionization modes.