Molecular fingerprinting of Staphylococcus aureus isolated from patients with osteomyelitis in Argentina and clonal distribution of the cap5(8) genes and of other selected virulence genes.

The molecular fingerprinting of a collection of 94 Staphylococcus aureus isolates from patients with osteomyelitis in Argentina was performed. Twenty-three SmaI pulsed-field gel electrophoresis (PFGE) types and 37 spa types were identified. The isolates were assigned to 23 sequence types (STs). The proportion of methicillin-resistant S. aureus (MRSA) isolates was significantly higher among cap5 S. aureus (35/61) compared with cap8 S. aureus (8/33) isolates (p = 0.0025). Twenty-four of the 94 isolates carried the lukS-PV/lukF-PV genes, which were significantly associated to cap5 [(23/38) compared with cap8 S. aureus isolates (1/32) (p = 0.0001)]. Forty of the 94 isolates carried genes of the egc locus (seg/sei). The distribution of seg/sei genes among isolates was related to certain clones. Isolates of the four agr types were found in the S. aureus collection. Whereas agr I isolates were evenly distributed among cap5 and cap8 S. aureus isolates (32/61 and 14/33, respectively), the agr II group was composed of 29 cap5 S. aureus isolates and agr III was composed of 16 cap8 S. aureus isolates. Two clones originally associated to animals (ST 188, 7 isolates and ST 1796, 5 isolates) were associated with chronic osteomyelitis and lack of capsular polysaccharide (CP) production. Loss of CP production remains the single factor among those investigated that is associated with chronic osteomyelitis.

A 15 year-review of peritoneal dialysis-related peritonitis: microbiological trends and patterns of infection in a teaching hospital in Argentina.

This study reports the infectious peritonitis rates in 44 patients on peritoneal dialysis in three different systems over the last 15 years, covering clinical outcomes, exit-site infections, tunnel infections, causative microorganisms, and the history of susceptibility of organisms causing peritonitis, in order to establish our center-specific selection of empiric therapy. Two microbiological procedures were herein used: method A, where 100 ml of dialysate were centrifuged and cultured in standard media and into blood-culture bottles; and method B, where 10 ml were directly injected into blood-culture bottles. Swabs from the exit-site or tunnel were taken when purulent drainage was observed. There were 96 episodes of peritonitis during 110.43 patient-years (0.87 episodes/patient-year). Sensitivity of method A was 96.88% (93/96 episodes) versus 81.25% (78/96) of method B (p=0.001). Gram stain sensitivity was 36.46%. The etiologic agents were 64 (56.64%) gram-positive cocci, 22 (19.47%) gram-negative fermentative rods, 20 (17.7%) gram-negative non fermentative rods, 5 (4.43%) yeasts, 1 (0.88%) micelial fungus, and 1 (0.88%) anaerobic rod. Fifty-five exit-site infections were documented (0.5 episodes/patient-year). Ceftazidime and imipenem showed excellent activity on gram-negative rods. There were 92.3% of methicillin-susceptible Staphylococcus aureus but only 33.3% of methicillin-susceptible coagulase-negative staphylococci; vancomycin was active against 100% of the gram-positive cocci. The clinical outcomes of peritonitis were 73 initial cure, 19 catheter removal and four related deaths. The empiric therapy in our center should be vancomycin plus ceftazidime or imipenem. Once the etiological agent and its susceptibility pattern are known, the deescalating therapy must be applied to avoid the emergence and spread of vancomycin-resistant microorganisms.

[Nonpuerperal breast abscess caused by Finegoldia magna]. / Absceso mamario no puerperal por Finegoldia magna.

Finegoldia magna is a species of strictly anaerobic gram-positive cocci, arranged in pairs, tetrads, and clusters. These organisms are components of the normal flora of the skin, gastrointestinal and genitourinary female tracts, and oral cavity. They are asaccharolytic and their major energy sources are aminoacids and peptones. The species is usually isolated in polymicrobial cultures from abscesses, soft tissue infections, bone and joints. In the case herein presented, F. magna was recovered in pure culture from a nonpuerperal breast abscess, which adds to the two reported cases in related literature. Species identification was performed by special potency disks, standard bacteriological anaerobic tests, and production of saccharolytic and proteolytic enzymes. Antimicrobial susceptibility testing was performed by using the epsilometric test. The agents assayed and MICs (microg/ml) values were: penicillin, 0.064; cephalotin, 1; metronidazole, 0.25; minocycline, < 0.016; azithromycin, 4; claritromycin, 2. We would like to highlight the importance of identifying anaerobic gram-positive cocci at species level, and of determining the antimicrobial susceptibility pattern, when they are isolated in pure culture from appropriate samples, as in the case presently reported.

Fungal peritonitis in patients on peritoneal dialysis: twenty five years of experience in a teaching hospital in Argentina.

Fungal peritonitis is a rare but serious complication of peritoneal dialysis. The aim of this study was to analyze peritonitis rates, associated factors, clinical course, microbiological aspects, therapeutic regimens, and outcome of patients with fungal peritonitis in the dialysis center of a teaching hospital over the last 25 years. A hundred and eighty three episodes of peritonitis were detected and microbiologically documented in 57 patients. Fungi were identified in eight episodes (4.37%) occurring in seven female patients. The fungal peritonitis rate was 0.06 episodes/patient-year. Gram and Giemsa stains were positive in five out of eight dialysate fluids. The causative microorganisms were: Candida albicans in five episodes, and Candida parapsilosis, Candida glabrata, and Neosartorya hiratsukae in the remaining three. Antibiotics were administered to all but one patient, within 3 months before fungal peritonitis was detected. All patients required hospitalization, and antifungal therapy was administered in all episodes. The Tenckhoff catheter was removed in seven out of eight fungal peritonitis. All patients recovered from the fungal episodes. In the group of patients studied, it is concluded that recent exposure to antibiotics and female sex, were strongly associated with the development of fungal peritonitis by yeasts. The peritonitis caused by the environmental filamentous fungus did not require antibiotic pressure. Direct microscopy of the dialysate pellet was extremely useful for the prompt management of the fungal episode. Fungal peritonitis preceded by multiple episodes of bacterial peritonitis always determined the definitive dropout of the patient from the peritoneal dialysis program. Patients with de novo yeast-related peritonitis could continue on the program.

Susceptibility trends of Bacteroides fragilis group isolates from Buenos Aires, Argentina.

The aim of this study was to analyze the susceptibility trends to seven antibiotics of Bacteroides fragilis group isolates based on three survey studies performed by the Committee of Anaerobic Bacteria between 1989 and 2002. Fifty three, 82 and 65 B. fragilis group isolates were collected during each period. The antimicrobial agents included were: ampicillin, ampicillin-sulbactam (2:1), cefoxitin, piperacillin, imipenem, clindamycin, and metronidazole. Minimal inhibitory concentrations (MICs) were determined according to the reference agar dilution method described by the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS). The most active antibiotics for B. fragilis and non-B. fragilis species throughout the three periods were: imipenem with 99.1 and 100% of activity, respectively, and metronidazole with 100% of activity. The susceptibility to ampicillin-sulbactam showed a decrease, from 100% to 90.3% and to 82.4 % in the last period, for both B. fragilis and non-B. fragilis species, respectively. The overall susceptibility rates for cefoxitin, piperacillin, and clindamycin were significantly different between B. fragilis and non-B. fragilis species (84.2% vs. 56.5%; 85.9% vs. 66.7% and 88.8% vs. 64.7%, respectively, p < 0.05). Cefoxitin was the antibiotic that showed more variations as regards periods and species. The susceptibility rates for clindamycin were low, about 60%, for non-B. fragilis species during the last two periods. The variations observed in the susceptibility patterns of the B. fragilis group isolates emphasize the need to continue monitoring the emergence of resistance in order to guide the election of the most appropriate antibiotic therapy scheme for anaerobic infections.

Methicillin-resistant Staphylococcus aureus strains in Buenos Aires teaching hospitals: replacement of the multidrug resistant South American clone by another susceptible to rifampin, minocycline and trimethoprim-sulfamethoxazole.

The aim of this study was to characterize methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from different infectious sites of hospitalized patients at two university hospitals. Fourteen isolates were analyzed by repetitive sequence based PCR (Rep-PCR), randomly amplified polymorphic DNA assay (RAPD-PCR), and pulsed-field gel electrophoresis (PFGE). We found that a prevalent clone of MRSA, susceptible to rifampin, minocycline, and trimethoprim/sulfamethoxazole (RIF(s), MIN(s), TMS(s)) was present in both hospitals in replacement of the multiresistant MRSA South American clone, previously described in these hospitals. The staphylococcal chromosomal cassette (SCCmec) type I element was detected in this new clone.

Detection of penicillin-binding proteins immunologically related to penicillin-binding protein 5 of Enterococcus hirae ATCC 9790 in Enterococcus faecium and Enterococcus faecalis.

Penicillin-binding protein (PBP) 5 of Enterococcus hirae ATCC 9790 belongs to the class of the high-molecular mass, low-affinity PBPs which have been correlated with penicillin resistance in most Enterococcus species. Polyclonal antibodies were raised against PBP 5 and used to detect immunologically related membrane proteins in E. faecium and E. faecalis strains. Several strains of both species were found to have a membrane protein of similar molecular mass to E. hirae PBP 5 which reacted with the antibodies. Some E. faecium strains did not react with antibodies but their derivatives with increased penicillin minimal inhibitory concentrations did. In some E. faecalis strains the lack of a PBP 5-related protein was associated with failure to select stable penicillin-resistant derivatives.

[Peritonitis in continuous ambulatory peritoneal dialysis. Microbiological and clinical evaluation]. / Peritonitis en diálisis peritoneal contínua ambulatoria. Evaluación microbiológica y clínica.

In 1976, Popovich et al. described a technique of peritoneal dialysis using bottled dialysate. Later Oreopoulos et al. modified the technique by using plastic bags. But peritonitis still is a major and potentially serious complication of peritoneal dialysis. We have evaluated a) microbiologic diagnostic methods for infectious peritonitis, b) incidence of etiologic agents, and c) the evolution during antimicrobial treatment. Eighteen patients with chronic renal failure of diverse causes were followed from initiation of the CAPD program since January 1981 until June 1988. There were 80 episodes of infectious peritonitis during 17 patient-years of dialysis with an overall incidence of peritonitis of 4.7 episodes/patient-year. The total volume centrifuged technique and culture of sediment showed a sensibility of 85% in 73 episodes where cultures were obtained. The 59.1% of episodes of peritonitis were caused by gram negative bacilli; 11.6% were due to Acinetobacter calcoaceticus and Gram positive cocci accounted for 37.3%. These results are different from those found in other countries because most of our patients had received antimicrobial agents which probably changed their body flora, some did not have manual ability, others were of bad hygienic habits and finally, all of them had frequent contact with hospital environment. The species most frequently isolated were coagulase negative staphylococci (12.8%), probably from patients' skin flora. (ABSTRACT TRUNCATED AT 250 WORDS)

[Bacteroides distasonis meningitis]. / Meningitis por Bacteroides distasonis.

A 50 year old woman while undergoing severe treatment for rheumatoid arthritis, developed anaerobic meningitis. The cerebrospinal fluid (CSF) sample was transported and cultivated aerobically and anaerobically. After 48 h at 37 degrees C the anaerobically incubated plate, the enriched fluid thioglycollate medium and the anaerobic culture medium yielded luxuriant growth of an anaerobic Gram negative bacillum. The biochemical and antimicrobial susceptibility patterns were consistent with those for Bacteroides distasonis. Most of the strains of the 5 species included in the Bacteroides fragilis group (B. fragilis, B. vulgatus, B. ovatus, B. thetaiotaomicron and B. distasonis) are resistant to penicillins, cephalosporins of first generation and aminoglycosides. Anaerobic polyresistant flora from an intraabdominal focus (chronic cholecystitis) might have been selected by treatment with gentamicin and cephalotin, and proliferated into meningeal dissemination. It is important that CSF from immunocompromised patients with acute or chronic pulmonary, intraabdominal or cranium-facial infectious processes be transported and cultured in aerobic and anaerobic conditions. These patients must be treated with an initial therapeutic scheme that includes an effective antibiotic for the anaerobic microorganism that may be involved.

Assessment in the diagnosis of male chronic genital tract infection.

Different methodologies have been proposed to interpret the microbiological findings associated with contaminating, indigenous microbiota of the anterior urethra. In order to solve the controversy related to the diagnosis of chronic seminal infections in asymptomatic young adults, the results applying Stamey and Meares' criteria were compared with those obtained when semen cultures were studied for significant bacteriospermia. A total of 218 consecutive asymptomatic male partners of infertile couples were evaluated by the four-specimen technique described by Stamey and Meares' with the addition of semen (SM). Infection was detected in 46% by SM, while semen cultures (SC) showed a prevalence of infection of 41%; 73 patients were positive by both criteria and 102 negative; 27 patients were positive by SM technique in prostate fluid while their semen cultures were negative; 16 patients had positive semen cultures and were considered negative by SM. The kappa statistic indicated a good degree of agreement between both methodologies (kappa = 0.61, z = 8.68, p < 0.001). The estimated risk of being considered negative attributable to the semen culture (27 patients) was 25% (attributable risk = gamma ac- = 0.2550), and of being considered positive attributable to the semen culture (16 patients) was 26% (gamma ac+ = 0.2579). The 95% confidence limits were estimated in 12 to 39%, and in 13 to 31%, respectively. In view of these results, to establish the diagnosis of chronic prostatitis, the addition of prostatic fluid or voided urine cultures after prostatic massage, must be performed. Semen culture confronted with first-voided urine avoid overestimating seminal infection.

Improvement of semen quality in infected asymptomatic infertile male after bacteriological cure.

Bacteriological etiology was investigated in 29 infected asymptomatic infertile males. The localization of the infection and the effect of a long term antibiotic therapy on semen parameters were evaluated. The most frequent etiological agent isolated was Enterococcus faecalis. Positive bacteriological culture was obtained in prostatic fluid in 16 patients and in semen in 13. Bacteriological cure was achieved in 24 cases and it was associated with improved seminal parameters: sperm concentration, viability and total motile sperm per ejaculate. In 5 patients without bacteriological cure there was no change in semen analysis after antibiotic therapy. In 45% of the infected patients there were less than 0.5 x 10(6)/ml seminal polymorphonuclear leukocytes. In view of these findings granulocyte concentration seems to be a poor marker to predict infection.

[Ovarian abscess due to Actinomyces sp. in absence of an intrauterine contraceptive device]. / Absceso ovárico por Actinomyces sp. en ausencia de un dispositivo intrauterino.

The disease caused by Actinomyces spp. is often of difficult diagnosis. Actinomyces spp. are anaerobic or microaerophilic non-spore-forming gram-positive rods that may reach, occasionally, the normal female genital tract. IUD and pessaries facilitate the access of the microorganisms to the pelvis. We report an unusual case of ovarian infection by Actinomyces sp. in a 41 year-old female without IUD, admitted at the Institute in November 1998, with persistent fever. She had had an early menopause 3 years before, and had received hormonal replacement therapy. Usual and unusual infections were discarded by microbiological and serologic studies. Abdominal ultrasonography showed a slight left pyelocalycial dilatation and a simple cyst in the left ovary; heart ultrasonography was normal. Gynecological examination showed an enlarged uterus, similar to an 8 week pregnancy, painless, and fixed anexial masses. The transvaginal ultrasonography showed uterine myomas, one of them of 42 mm in the isthmus region, large ovaries, cystic, with acoustic shadows, and the left one with a septum. The preoperative diagnosis was infected bilateral cystic teratoma. The procedure was an exploratory laparotomy, followed by a bilateral salpingo-oophorectomy. The specimen studies showed an endometrioma with calcium deposits in the wall of the right ovary, and an abscess in the left ovary, also with calcification of the wall. The sample from the left abscess developed Actinomyces sp. After surgery, and treatment with penicillin, the fever disappeared. It is important to remark that the ovarian infection by Actinomyces sp. can also occur in patients without an IUD or a pessary; it might cause anexial images that can be interpreted as a tumour, inducing to erroneous diagnosis and treatment.

[Use of sebacic acid for identifying anaerobic bacteria using gas phase chromatography]. / Uso del ácido sebácico en la identificación de bacterias anaerobias por cromatografía en fase gaseosa (CGL).

Volatile short-chain fatty acids formed as end products of metabolism of 25 anaerobic bacterial strains isolated from different clinical specimens and collections were analysed by GLC. Strains were previously identified using microscopic, cultural and biochemical tests. Gas-chromatograms of acids (volatile fatty acid profiles) were obtained with a sebacic acid packed column. The potential application of the GLC technique for the rapid and accurate identification of these organisms is discussed.

Susceptibility of Enterococcus faecalis to twelve antibiotics, time-kill assays, and high-level aminoglycoside resistance in a university hospital in Argentina.

A total of 201 Enterococcus faecalis strains isolated from different body sites were tested to (i) establish their antibiotic susceptibility pattern; (ii) determine the percentage of strains highly resistant (MIC greater than 2,000 micrograms/ml) to five aminoglycosides and (iii) know if the combination of penicillin or ampicillin plus an aminoglycoside is reliably synergistic for the strains with low-level resistance (MICs ranged from the break point of susceptibility for each aminoglycoside to 2,000 micrograms/ml). Erythromycin exhibited very poor activity with MIC90 greater than 128 micrograms/ml. Pefloxacin and norfloxacin had intermediate activity, inhibiting 50% of isolates at 4 micrograms/ml and 90% at 8 micrograms/ml. Trimethoprim-sulfamethoxazole (1:20) inhibited 94% of isolates at less than or equal to 2 micrograms/ml and 87.6% at less than or equal to 0.5 microgram/ml. Ampicillin, penicillin and piperacillin were the most potent agents studied. Ninety percent of strains were inhibited at 1 microgram/ml of ampicillin and 4 micrograms/ml of penicillin and piperacillin. The E. faecalis isolates were relatively or totally resistant to the aminoglycosides. Ninety six (47.8%) were highly resistant at least to one of them. High level resistance to streptomycin was found in 47.3% of all strains and was the most frequent resistance encountered; amikacin highly resistant strains were the less common and accounted for 4.5%. Low-level resistance to the aminoglycosides ranged from 50.2% (for streptomycin) to 94.5% (for amikacin). Thirty one E. faecalis isolates were selected for 24-time kill-assays. There was a good correlation between resistance to penicillin or ampicillin aminoglycoside synergy in all but 3 strains which were highly resistant. Among the strains with low-level resistance to the aminoglycosides, there was no synergy in 37 (63.8%) of 58 killing assays with each of the aminoglycosides combined with penicillin. These results demonstrate that the susceptibility to 2,000 micrograms/ml of the aminoglycoside does not assure the penicillin or ampicillin aminoglycoside synergism.

["In vitro" activity of ten antimicrobial agents against anaerobic bacteria. A collaborative study, 1999-2002]. / Actividad "in vitro" de 10 antimicrobianos frente a bacterias anaerobias. Estudio multicéntrico, 1999-2002.

The antimicrobial activity of ampicillin, ampicillin-sulbactam, cefoxitin, ceftriaxone, imipenem, piperacillin, piperacillin-tazobactam, clindamycin, metronidazole, and azitromycin was assesed against 166 strains of anaerobic bacteria recovered from eight hospitals in Buenos Aires. The strains studied were Bacteroides fragilis group (65), Fusobacterium spp. (26), Prevotella spp. (21), Porphyromonas spp. (10), Clostridium difficile (10), other clostridia (12), and gram-positive cocci (22). The MICs were determined by the agar dilution method according to NCCLS document M11-A5. Metronidazole and piperacillin-tazobactam were the most active antimicrobial agents tested and exhibited MIC90 values of < or = 2 microg/ml and < or = 4 microg/ml against gram-negative organisms, and < or = 2 microg/ml, and < or = 8 microg/ml against gram-positive organisms, respectively. Among beta-lactams the activity against gram-negative rods was in the following order: imipenem > piperacillin > cefoxitin > ceftriaxone > ampicillin. Among the gram-positive bacteria the decreased activity was: piperacillin > imipenem > cefoxitin > ceftriaxone > ampicillin. The majority of the species studied showed different degrees of resistance to clindamycin and azitromycin. Nevertheless, 90% of Fusobacterium nucleatum and Porphyromonas spp. isolates were inhibited by 0.125 mg/ml of clindamycin and azitromycin, respectively.

[Consensus on antimicrobial sensitivity tests in gram-positive cocci. Subcommittee on Antimicrobials, SADEBAC (Argentinian Society of Clinical Bacteriology), Argentinian Association of Microbiology]. / Consenso sobre las pruebas de sensibilidad a los antimicrobianos en cocos gram-positivos.

Antimicrobial susceptibility testing is mainly performed in Argentina by disk diffusion method, following National Committee for Clinical Laboratory Standards (NCCLS) recommendations. We worked out new recommendations for the reporting and interpretation of this test when dealing with gram-positive cocci, in accordance to local trends and epidemiology. General considerations for performing the diffusion assay, quality control, and an update on susceptibility testing for gram-positive cocci are reported in this first document. The present update should be considered as a group of recommendations summarized by Argentinean experts and as the result of a consensus meeting coordinated by the Subcomisión de Antimicrobianos of the Sociedad Argentina de Bacteriología Clínica (Asociación Argentina de Microbiología). Experts in antimicrobial agents were convened in order to prepare this final document. These recommendations take into account local needs, affordability and availability to be used in current practice, tending to contribute to the correct antimicrobial treatment election, according to the particular microorganism and the infection sites.

Genotypic identification of methicillin-resistant coagulase-negative staphylococci by polymerase chain reaction.

A rapid method for the detection of methicillin resistance in staphylococci was developed. The method was based on the polymerase chain reaction (PCR) and primers that targeted the internal region of the coding frame of the mec gene. The amplification reaction was carried out with crude cell lysates as a source of target DNA and provided data in less than 5 h. Seventy-four isolates of coagulase-negative staphylococci were tested by PCR, DNA hybridization with a probe derived from the mec gene, and an agar dilution susceptibility assay. PCR results showed a 100% correlation with the susceptibility assay carried out with high inocula (10(8) CFU) and incubation at 32 degrees C for 48 h. PCR was more sensitive and specific than DNA hybridization in detecting methicillin resistance in coagulase-negative staphylococci. The former technique identified the mec gene in all the strains which were phenotypically resistant but which did not hybridize with the probe. Identification of methicillin-resistant strains by PCR offers a very specific, sensitive, and rapid alternative to traditional susceptibility tests and DNA hybridization as a guide for the treatment of infections caused by staphylococci.

Broth disk elution method for anaerobic bacteria: a collaborative study to assess its reliability for clinical purposes.

A collaborative study involving seven laboratories was undertaken to evaluate the reproducibility and the reliability of the broth disk elution test against anaerobic bacteria by comparing with the reference agar dilution method. A two breakpoint broth test was also evaluated. Assays were performed using the same testing conditions (i.e. medium, temperature, atmosphere and incubation time). One hundred Gram-negative and Gram-positive clinical isolates were initially studied. Overall agreement of 98.5% and 97.5%, were found for disk elution and the two breakpoint tests, respectively. In order to assess the reliability of the disk elution test, two different lots (LOT1 and LOT2) of disks of piperacillin and clindamycin were selected, to obtain two final concentrations after dilution (10 and 60 mg/mL; 1 and 4 mg/mL, respectively). Two hundred and eighty assays were performed against one strain of both Bacteroides fragilis(piperacillin MIC, 8.0 mg/mL; clindamycin MIC, <0.5 mg/mL) and Bacteroides thetaiotaomicron(piperacillin MIC, 16.0 mg/mL; clindamycin MIC, <0.5 mg/mL). With LOT 1, considering both species and both antibiotics, the agreement among six laboratories ranged from 85% to 100% (P > 0.05) with the higher concentration. Overall agreement among all laboratories was 91%. No optimal agreement (>90%) for clindamycin-Bacteroides thetaiotaomicron using the LOT1 (77%) was found. Since this finding was not observed with LOT2 (100% agreement), discrepancies were attributed to variation between lots. Overall agreement with LOT2 was 100% for all centres. The present study indicates that the broth disk elution method proved to be a reliable and suitable alternative for routine susceptibility testing for anaerobic bacteria, as a resistance screening method for clinical purposes.