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The purpose of this study was to determine the carriage rate, susceptibility pattern, and serotype distribution of Streptococcus pneumoniae in the nasopharynx of children younger than 2 years old in Lima, Peru. A total of 666 children were evaluated during 3 periods, 1997, 2001, and 2003. The overall pneumococcal carrier rate was 41%. Reduced susceptibility to penicillin was found in 5% (4/75) of isolates in 1997, 20% (15/75) in 2001, and 37% (40/109) in 2003. Reduced susceptibility to ceftriaxone was found in 12% of isolates in 2003. Serogroups 6, 19, 23, 15, and 14 accounted for 68% of all the isolates and for 81% of the penicillin-nonsusceptible strains. Only 65% of the isolated strains had serogroups found in the 7-valent conjugate pneumococcal vaccine. This highlights the importance of regional surveillance studies for effective vaccine strategies and treatment protocols.

Increasing resistance in Pseudomonas aeruginosa to multiple antibiotics has been observed and is posing therapeutic dilemmas. Antibiotic utilization is one factor that has been associated with the emergence of antimicrobial resistance. We examined the overall and specific antimicrobial use in relation to changes in susceptibility patterns in P. aeruginosa. Regression analysis was performed to explore the relationships between annual antibiotic use and the incidence of resistant P. aeruginosa. There were statistically significant relationships between increasing anti-pseudomonal cephalosporin and levofloxacin use and the increasing incidence of ciprofloxacin resistant P. aeruginosa. However, there was not an association between other fluoroquinolone or overall fluoroquinolone use and this change. In addition, there was no association between increasing anti-pseudomonal cephalosporin use and cefepime resistant P. aeruginosa. No statistical relationship was seen with overall antibiotic use and the development of resistance in P. aeruginosa, suggesting that the development of resistance is associated with the use of individual agents, rather than overall antibiotic consumption.

Isoniazid (INH) is a central component of drug regimens used worldwide to treat tuberculosis. Previous studies have identified resistance-associated mutations in katG , inhA , kasA , ndh , and the oxyR-ahpC intergenic region. DNA microarray-based experiments have shown that INH induces several genes in Mycobacterium tuberculosis that encode proteins physiologically relevant to the drug's mode of action. To gain further insight into the molecular genetic basis of INH resistance, 20 genes implicated in INH resistance were sequenced for INH resistance-associated mutations. Thirty-eight INH-monoresistant clinical isolates and 86 INH-susceptible isolates of M. tuberculosis were obtained from the Texas Department of Health and the Houston Tuberculosis Initiative. Epidemiologic independence was established for all isolates by IS 6110 restriction fragment length polymorphism analysis. Susceptible isolates were matched with resistant isolates by molecular genetic group and IS 6110 profiles. Spoligotyping was done with isolates with five or fewer IS 6110 copies. A major genetic group was established on the basis of the polymorphisms in katG codon 463 and gyrA codon 95. MICs were determined by the E-test. Semiquantitative catalase assays were performed with isolates with mutations in the katG gene. When the 20 genes were sequenced, it was found that 17 (44.7%) INH-resistant isolates had a single-locus, resistance-associated mutation in the katG , mabA , or Rv1772 gene. Seventeen (44.7%) INH-resistant isolates had resistance-associated mutations in two or more genes, and 76% of all INH-resistant isolates had a mutation in the katG gene. Mutations were also identified in the fadE24 , Rv1592c , Rv1772 , Rv0340 , and iniBAC genes, recently shown by DNA-based microarray experiments to be upregulated in response to INH. In general, the MICs were higher for isolates with mutations in katG and the isolates had reduced catalase activities. The results show that a variety of single nucleotide polymorphisms in multiple genes are found exclusively in INH-resistant clinical isolates. These genes either are involved in mycolic acid biosynthesis or are overexpressed as a response to the buildup or cellular toxicity of INH.

Vancomycin and daptomycin MICs from 161 isolates of methicillin-resistant Staphylococcus aureus (MRSA) were compared using commercial and in-house broth microdilution, Etest, and common automated methods. Vancomycin Etest MICs were higher than those of other methods, whereas the MICs for daptomycin testing were comparable. Vancomycin MICs vary depending on the testing methodology.

Mycobacterium tuberculosisandMycobacterium aviumare facultative intracellular pathogens that are able to survive and replicate in mononuclear phagocytes. Human complement component C3 has previously been shown to mediate attachment and phagocytosis of these bacteria by mononuclear phagocytes. In this study, a C3 ligand affinity blot protocol was used to identify a 30-kDa C3-binding protein inM. tuberculosisandMycobacterium smegmatisand a 31-kDa C3-binding protein inM. avium. The C3-binding proteins inM. tuberculosisandM. aviumlocalized to the cell membrane fraction and partitioned to the detergent fraction during Triton X-114 phase partitioning. The C3-binding protein fromM. tuberculosiswas partially purified using a cation exchange column and was shown to bind concanavalin A. The N terminus and an internal fragment of the partially purified C3-binding protein were subjected to amino acid sequence analysis. The resulting amino acid sequences matched theM. tuberculosisheparin-binding hemagglutinin (HbhA) protein. Recombinant full-length HbhA and the C terminus of HbhA fused to maltose-binding protein, but not recombinant HbhA lacking the C-terminal region, bound human C3. Recombinant full-length HbhA coated on polystyrene beads, was found to enhance the adherence and/or phagocytosis of the coated beads to J774.A1 cells in both the presence and absence of human serum. The presence of complement-sufficient serum increased the adherence of the HbhA-coated beads to the J774.A1 cells in a C3-dependent manner. If HbhA within the bacterial cell membrane functions similarly to isolated HbhA, this protein may enhance the adherence and phagocytosis ofM. tuberculosisandM. aviumto mononuclear phagocytes through the binding of C3 and interaction with C3 receptors on mononuclear phagocytes.

After decades of decline in developed countries, there was a resurgence of tuberculosis in the mid-1980s accompanied by increased recognition that this infectious disease has long remained a major public health problem at the global level. New methods from molecular biology, in particular DNA 'fingerprinting' (of Mycobacterium tuberculosis), made it clear that current transmission and recent infection (in contrast to reactivation of earlier, latent infection) were much more significant than previously believed. Studies of tuberculosis outbreaks using these new tools pointed to complex networks through which infection was spreading and highlighted the need for new approaches to outbreak investigation and disease control. In the study reported here a new approach--combining methods from molecular biology, epidemiology and network analysis--was used to examine an outbreak of tuberculosis in Houston, Texas. Initial investigation using conventional strategies revealed few contacts among 37 patients with identical (six-band) DNA (IS6110-based) fingerprints but subsequent research uncovered over 40 places (including many gay bars) to which patients in this outbreak could be linked. Network methods were used to reconstruct an outbreak network and to quantify the relative importance (here, 'betweenness' centrality) of different actors (persons and places) playing a role in the outbreak. The multidisciplinary work provides the basis for a new approach to outbreak investigation and disease control.

Limited data suggest that measures to reduce tuberculosis transmission should be based on locations rather than on personal contacts. Molecular epidemiologic methods (analysis of IS6110 patterns, spoligotypes, variable numbers of tandem DNA repeats, and automated DNA sequence data) identified a cohort of 48 persons who were infected with progeny of the same Mycobacterium tuberculosis strain. Epidemiologic investigation documented that a large proportion of the patients were gay white human immunodeficiency virus-positive men. Most practiced barhopping, an activity that involved patronizing many bars in the same neighborhood each night. Few subjects were directly linked to more than 1 or 2 other persons by conventional investigation methods, which shows that the transmission dynamics were unusually complex compared with most previously described episodes of strain spread. The data support the concept that identification of locations where pathogen dissemination likely occurs may provide additional strategies for targeted tuberculosis control.

A multicenter study was conducted to assess the interlaboratory reproducibility of broth microdilution testing of the more common rapidly growing pathogenic mycobacteria. Ten isolates (four Mycobacterium fortuitum group, three Mycobacterium abscessus , and three Mycobacterium chelonae isolates) were tested against amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, imipenem, sulfamethoxazole, and tobramycin ( M. chelonae only) in four laboratories. At each site, isolates were tested three times on each of three separate days (nine testing events per isolate) with a common lot of microdilution trays. Agreement among MICs (i.e., mode ± 1 twofold dilution) varied considerably for the different drug-isolate combinations and overall was best for cefoxitin (91.7 and 97.2% for one isolate each and 100% for all others), followed by doxycycline, amikacin, and ciprofloxacin. Agreement based on the interpretive category, using currently suggested breakpoints, also varied and overall was best for doxycycline (97.2% for one isolate and 100% for the rest), followed by ciprofloxacin and clarithromycin. Reproducibility among MICs and agreement by interpretive category was most variable for imipenem. Based on results reported from the individual sites, it appears that inexperience contributed significantly to the wide range of MICs of several drugs, especially clarithromycin, ciprofloxacin, and sulfamethoxazole. New interpretive guidelines are presented for the testing of M. fortuitum against clarithromycin; M. abscessus and M. chelonae against the aminoglycosides; and all three species against cefoxitin, doxycycline, and imipenem.

We read with great interest the article by Shelburne et al. ([4][1]) describing two different clones of multidrug-resistant (MDR) Acinetobacter baumannii with distinct clinical outcomes. In that report, carried out in a university hospital in Texas, the authors detected the presence of two major

Plasmodium falciparum typically manifests as a severe, symptomatic infection due to the high parasitemia and increased adherence of infected cells to blood vessel endothelium. However, there are case reports of asymptomatic, indolent P falciparum infections that reactivated months or years later, even though P falciparum lacks a hypnozoite form. We report a case of a 56 year old …

The increasing proportion of Streptococcus pneumoniae isolates with reduced susceptibility to penicillin has created an urgent need for therapeutic alternatives to some β-lactam agents. Clindamycin is an antimicrobial agent with excellent bioavailability after oral administration which has been considered for the therapy of community-acquired pneumococcal otitis media. Using the Etest methodology, we have studied the in vitro susceptibility of 59 erythromycin-resistant strains of S. pnuemoniae to clindamycin, penicillin, trimethoprim-sulfamethoxazole, and rifampin. The study also addressed the impact of the susceptibility test medium [mueller-Hinton (MH) vs IsoSensitest (Iso), both 5% blood supplement] on the results. A total of 20 isolates (37%) displayed constitutive clindamycin resistance on Iso blood agar, compared with only 11 (22%) on MH blood agar. The remaining nine strains found to be clindamycin susceptible on MH manifested resistance only with erythromycin induction. Resistance to penicillin, rifampin, and trimethoprim-sulfamethoxazole in erythromycin-resistant isolates was 83%, 2%, and 85%–89% (medium dependent), respectively. These results indicate that the choice of susceptibility test medium affects the expression (constitutive or inducible) of macrolide-lincosamide-streptogramin (MLS) resistance in S. pneumoniae . In addition, the common assumption that erythromycin resistance in S. pneumoniae implies clindamycin resistance may need to be reconsidered and routine susceptibility tests (including induction if MH medium is used) should be considered for MLS-class drugs.

Etest (AB BIODISK, Solna, Sweden) is a precise MIC method and the practical method of choice for the susceptibility testing of many fastidious organisms, including rapidly growing mycobacteria. Methods recommended by the National Committee for Clinical Laboratory Standards for the susceptibility testing of Mycobacterium tuberculosis include the Bactec (Becton Dickinson, Sparks, Md.) broth and agar proportion methods. A comparison of Etest with the Bactec broth method for testing the susceptibility of M. tuberculosis to four first-line antituberculous agents demonstrated equivalent interpretive results for 100% of the isolates tested. Agreements with agar proportion MICs, within +/-2 log2 dilutions, were 90, 93, 100, and 94% for ethambutol, isoniazid, rifampin, and streptomycin, respectively. Etest MICs were easily read within 5 to 10 days of inoculation. Preparation of the inoculum with a turbidity equivalent to a McFarland 3.0 standard prepared from growth on an agar surface and with a broth with a Bactec growth index of > 999 yielded equivalent results. Clinical isolates for which the MICs were reproducible were also identified as possible quality control strains. The Etest method appears to be an alternative method for testing the susceptibility of M. tuberculosis isolates to the four most commonly used therapeutic agents.

Methods currently used for susceptibility testing of Mycobacterium, including agar dilution and the Bactec radiometric method, are based on the proportion technique using a single critical concentration of antibiotic. Preliminary studies were conducted with Mycobacterium species by using the Etest method, a gradient minimum inhibitory concentration technique, well described for susceptibility testing of other fastidious and slow-growing organisms. Excellent correlation was demonstrated between Etest and agar dilution with rifampin-susceptible and -resistant isolates of M. tuberculosis and ciprofloxacin-resistant and -susceptible isolates of Mycobacterium avium-intracellulare.

We examined the in vitro activity of PD 127391, an investigational fluoroquinolone antibacterial agent, against staphylococci (including methicillin-resistant Staphylococcus aureus), enterococci (including beta-lactamase-producing and highly gentamicin-resistant isolates), and streptococci. The compound was active against all organisms tested and compared favorably with antimicrobial agents routinely used to treat infections with these organisms. On the basis of MICs for 90% of the strains tested, PD 127391 was 32-fold more active against all staphylococci, 16-fold more active against methicillin-resistant S. aureus, 8-fold more active against all streptococci, and 4-fold more active against all enterococci than ciprofloxacin. PD 127391 was shown to be more active than sparfloxacin, which in turn was shown to be more active than ciprofloxacin, against these gram-positive cocci. PD 127391 shows promise for the treatment of infections with gram-positive cocci, including organisms which are resistant to other commonly used antimicrobial agents.

In Staphylococcus aureus, beta-lactamase (Bla) is typically associated with transducible plasmids that also encode metal resistance or with the more recently described conjugative, gentamicin resistance plasmids. The beta-lactamase gene of Enterococcus faecalis, which has been shown to be highly homologous to bla from the S. aureus plasmid p1258, is also encoded on conjugative, gentamicin resistance plasmids. To determine if the enterococcal Bla-encoding plasmids are similar to those from staphylococci, Bla-encoding plasmids from both species were compared by restriction endonuclease digestion followed by hybridization to Bla and gentamicin resistance gene probes and to one of the enterococcal Bla-encoding plasmids. Although similarities were noted in the restriction endonuclease digestion patterns among the transducible staphylococcal plasmids (pI524, pI258, and pII147) and among the conjugative Gmr staphylococcal plasmids, neither of these restriction patterns was similar to those of the enterococcal plasmids. Although the enterococcal plasmids showed extensive similarities by hybridization, the staphylococcal plasmids showed little or no similarity to the enterococcal plasmids except for bla and the gentamicin resistance genes. Whether enterococci acquired a different type of Bla-encoding plasmid or whether bla was acquired by transposition or by homologous recombination into enterococcal plasmids is unknown.

A 42-year-old man, with a history of immunoglobulin A nephropathy, underwent a living-related kidney transplant. Allograft function progressively deteriorated secondary to chronic rejection and recurrence of IgA nephropathy, and he returned to peritoneal dialysis after 5 years of the transplant. Fifteen months after the discontinuation of immunosuppressive therapy, Eschericia coli peritonitis developed, which was treated with ceftazidime intraperitoneally; he received fluconazole as prophylactic antifungal therapy during this period. After completing his course of treatment, abdominal pain occurred with an increased peritoneal fluid white blood cell count. Peritoneal fluid cultures were negative. He received broad-spectrum antibiotics and fluconazole with no appreciable response. After removal of the Tenckoff catheter, peritoneal fluid cultures grew a zygomycete. The patient was treated with liposomal amphotericin B (AmBisome) intravenously for 6 weeks. He had episodes of recurrent intraabdominal abscesses requiring surgical drainage and antibiotics. A second course of liposomal amphotericin B was administered for histopathologic evidence of filamentous fungal recurrence. After 5 months, the patient remains well without any evidence of infection.

Five infants from a day care center developed severe diarrhea associated with enteropathogenic Escherichia coli O114:nonmotile (EPEC O114:NM) and required hospitalization. Five additional cases of diarrhea associated with EPEC O114:NM subsequently occurred, four in hospital contacts of the patients and one in a household contact. Biochemically, all EPEC O114:NM isolates were sorbitol nonfermenters. All isolates produced low concentrations of cytotoxin with a mean of 10(1.23) CD50/mg of protein. Cytotoxin was not neutralized with antibody to Shiga-like toxin I or II. Heat-labile and heat-stable enterotoxins were not present by gene probe analysis. Stool isolates from 9 of 10 hospitalized infants were positive for EPEC adherence factor by colony blot DNA probe analysis. The severity of the disease, sorbitol nonfermentation, and presence of enteroadherence are unusual features of this organism.

We tested the activity of LY146032 (LY) against 57 strains of enterococci collected from Chile, Thailand, and the United States. Some of the strains were resistant to high levels of gentamicin or streptomycin (or both), and two produced beta-lactamase (Bla+). MICs of LY ranged from 0.5 to 8 micrograms/ml, and MBCs ranged from 1 to 64 micrograms/ml. In time-kill assays, a 2 to 3 log10 killing effect was observed with LY against two Bla+ strains of Streptococcus (Enterococcus) faecalis and against three strains that were highly resistant to streptomycin and gentamicin. Synergism was demonstrated with LY and streptomycin against a Bla+ strain lacking high-level streptomycin resistance. These in vitro results suggest that LY should be studied further for possible use in treatment of enterococcal infections.

En utilisant une sonde d'ADN de 17 KJ on a etudie l'incidence de l'infection par E. coli entero-invasif chez des etudiants voyageant au Mexique. On a ainsi determine les caracteristiques biochimiques, la sensibilite aux bactericides, les serotypes et les formes plasmidiques de ces souches

The pathogenesis of the wide-spectrum human disease caused by Salmonella species is poorly understood. Cytotoxin production by other enteric pathogens has been increasingly investigated recently, and data are accumulating regarding the role of cytotoxins in enteric infections and hemolytic uremic syndrome. We studied the cytotoxic activity of 131 Salmonella strains of the major serotypes, including 94 strains of Salmonella enteritidis, 12 strains of Salmonella typhi, and 25 strains of Salmonella choleraesuis. Cytotoxicity was quantitatively determined in sonic extracts by a [3H]thymidine-labeled HeLa cell assay. All Salmonella strains examined showed some degree of cytotoxic activity. The geometric means +/- standard deviations of the amounts of cytotoxin produced (50% cytotoxic dose per milligram of bacterial protein) were 27 +/- 2 for S. typhi, 65 +/- 2 for S. enteritidis, and 117 +/- 2 for S. choleraesuis. Analysis of variance showed that the differences in cytotoxin production by the three species were significant (P less than 0.001). No significant differences were found between stool isolates and invasive strains of the same species. Neutralization studies showed that the cytotoxins produced by all Salmonella strains were immunologically distinct from Shiga toxin and the closely related Shiga-like toxins produced by Escherichia coli. DNA hybridization studies with DNA probes for Shiga-like toxins of types I and II showed no hybridization. In each species the cytotoxin was heat labile and sensitive to trypsin treatment, which indicated that its active component was probably protein in nature. Upon ultrafiltration with Amicon membranes and gel filtration chromatography, cytotoxic activity was found in the molecular weight range of 56,000 to 78,000. Our findings indicate that salmonellae produce cytotoxin(s) that may play a role in the manifestations of the various species.

A second strain of enterococcus (PA) producing beta-lactamase (Bla+ phenotype) was compared with the previously reported Bla+ enterococcus, strain HH22. As with the original strain, there was a marked inoculum effect when PA was tested with penicillin, ampicillin, and piperacillin; no difference was noted with methicillin, cephalothin, imipenem, or vancomycin; the difference with ticarcillin was intermediate. High-level gentamicin resistance (Gmr) transferred from PA to an enterococcal recipient strain at a frequency approximately 100-fold lower than for HH22; all Gmr transconjugants from both strains were Bla+, but only PA showed linkage of Gmr and Bla+ with transfer of resistance to streptomycin, tetracycline, and chloramphenicol. EcoRI digestion of plasmid DNA from Gmr Bla+ transconjugants showed no similarities between the two strains. A 5.1-kilobase EcoRI Bla+-encoding fragment derived from HH22 was cloned into an Escherichia coli cloning vector and shown to hybridize to a 10.2-kilobase EcoRI fragment derived from PA; both fragments hybridized to an 840-base-pair staphylococcal Bla+ gene probe. These data indicate that the penicillinases are similar but encoded on different or differently arranged plasmids. The fact that both are transferable emphasizes the potential for this new streptococcal resistance determinant to disseminate.