Soraya S. Andrade

Rapid Detection and Identification of Metallo-β-Lactamase-Encoding Genes by Multiplex Real-Time PCR Assay and Melt Curve Analysis

ABSTRACT Metallo-β-lactamase enzymes (MβL) are encoded by transferable genes, which appear to spread rapidly among gram-negative bacteria. The objective of this study was to develop a multiplex real-time PCR assay followed by a melt curve step for rapid detection and identification of genes encoding MβL-type enzymes based on the amplicon melting peak. The reference sequences of all genes encoding IMP and VIM types, SPM-1, GIM-1, and SIM-1 were downloaded from GenBank, and primers were designed to obtain amplicons showing different sizes and melting peak temperatures (Tm). The real-time PCR assay was able to detect all MβL-harboring clinical isolates, and the Tm-assigned genotypes were 100% coincident with previous sequencing results. This assay could be suitable for identification of MβL-producing gram-negative bacteria by molecular diagnostic laboratories.

Metallo-beta-lactamase detection: comparative evaluation of double-disk synergy versus combined disk tests for IMP-, GIM-, SIM-, SPM-, or VIM-producing isolates.

ABSTRACT The emergence of metallo-β-lactamase (MBL)-producing isolates is a challenge to routine microbiology laboratories, since there are no standardized methods for detecting such isolates. The aim of this study was to evaluate the accuracy of different phenotypic methods to detect MBL production among Pseudomonas spp., Acinetobacter spp., and enterobacterial isolates, including GIM, IMP, SIM, SPM, and VIM variants. A total of 46 genetically unrelated Pseudomonas aeruginosa, Pseudomonas putida, Acinetobacter sp., and enterobacterial strains producing distinct MBLs were tested. Nineteen strains were included as negative controls. The inhibition of bacterial growth and β-lactam hydrolysis caused by MBL inhibitors (IMBL) also were evaluated. The isolates were tested for MBL production by both a double-disk synergy test (DDST) and a combined disk assay (CD) using imipenem and ceftazidime as substrates in combination with distinct IMBL. One hundred percent sensitivity and specificity were achieved by DDST using 2-mercaptopropionic acid in combination with ceftazidime and imipenem for the detection of MBL production among P. aeruginosa and Acinetobacter species isolates, respectively. The CD test showed the same results for detecting MBL-producing enterobacteria by combining imipenem and EDTA, with a 5.0-mm-breakpoint increase in the size of the inhibition zone. Our results indicate that both phenotypic methods to detect MBL-producing isolates should be based on the genera to be tested, regardless of the enzyme produced by such isolates, as well as on the local prevalence of MBL producers.

Increased resistance to first-line agents among bacterial pathogens isolated from urinary tract infections in Latin America: time for local guidelines?

Emerging resistance phenotypes and antimicrobial resistance rates among pathogens recovered from community-acquired urinary tract infections (CA-UTI) is an increasing problem in specific regions, limiting therapeutic options. As part of the SENTRY Antimicrobial Surveillance Program, a total of 611 isolates were collected in 2003 from patients with CA-UTI presenting at Latin American medical centers. Each strain was tested in a central laboratory using Clinical Laboratory Standard Institute (CLSI) broth microdilution methods with appropriate controls. Escherichia coli was the leading pathogen (66%), followed by Klebsiella spp. (7%), Proteus mirabilis (6.4%), Enterococcus spp. (5.6%), and Pseudomonas aeruginosa (4.6%). Surprisingly high resistance rates were recorded for E. coli against first-line orally administered agents for CA-UTI, such as ampicillin (53.6%), TMP/SMX (40.4%), ciprofloxacin (21.6%), and gatifloxacin (17.1%). Decreased susceptibility rates to TMP/SMX and ciprofloxacin were also documented for Klebsiella spp. (79.1 and 81.4%, respectively), and P. mirabilis (71.8 and 84.6%, respectively). For Enterococcus spp., susceptibility rates to ampicillin, chloramphenicol, ciprofloxacin, and vancomycin were 88.2, 85.3, 55.9, and 97.1%, respectively. High-level resistance to gentamicin was detected in 24% of Enterococcus spp. Bacteria isolated from patients with CA-UTI in Latin America showed limited susceptibility to orally administered antimicrobials, especially for TMP/SMX and fluoroquinolones. Our results highlight the need for developing specific CA-UTI guidelines in geographic regions where elevated resistance to new and old compounds may influence prescribing decisions.

Advances in the microbiological diagnosis of sepsis.

Accurate diagnostic tests are essential for the correct identification of etiologic agents causing sepsis. Conventional microbiology cultures are time consuming and may even yield negative results in many cases of septic shock. In this manner, molecular-based technologies are emerging as promising tests for use into routine clinical laboratories. In this review, we discuss current available molecular methods for bacteremia diagnosis in adult and pediatric patients with suspected or confirmed sepsis. Results of studies using polymerase chain reaction, real-time polymerase chain reaction, and complementary DNA/oligonucleotide microarrays are described and discussed into the current scenario. These new methodologies are able to detect even small amounts of bacterial DNA directly from blood specimens and show increased sensitivity and specificity for detecting many infectious agents associated with sepsis. Despite some limitations presented by nucleic acid-based techniques, these genotypic tests can be useful along with traditional microbiology diagnostics.

Antimicrobial susceptibility patterns of unusual nonfermentative gram-negative bacilli isolated from Latin America: report from the SENTRY Antimicrobial Surveillance Program (1997-2002)

The antimicrobial susceptibility of 176 unusual non-fermentative gram-negative bacilli (NF-GNB) collected from Latin America region through the SENTRY Program between 1997 and 2002 was evaluated by broth microdilution according to the National Committee for Clinical Laboratory Standards (NCCLS) recommendations. Nearly 74% of the NF-BGN belonged to the following genera/species: Burkholderia spp. (83), Achromobacter spp. (25), Ralstonia pickettii (16), Alcaligenes spp. (12), and Cryseobacterium spp. (12). Generally, trimethoprim/sulfamethoxazole (MIC50, < 0.5 microg/ml) was the most potent drug followed by levofloxacin (MIC50, 0.5 microg/ml), and gatifloxacin (MIC50, 1 microg/ml). The highest susceptibility rates were observed for levofloxacin (78.3%), gatifloxacin (75.6%), and meropenem (72.6%). Ceftazidime (MIC50, 4 microg/ml; 83.1% susceptible) was the most active beta-lactam against B. cepacia. Against Achromobacter spp. isolates, meropenem (MIC50, 0.25 microg/ml; 88% susceptible) was more active than imipenem (MIC50, 2 microg/ml). Cefepime (MIC50, 2 microg/ml; 81.3% susceptible), and imipenem (MIC50, 2 microg/ml; 81.3% susceptible) were more active than ceftazidime (MIC50, >16 microg/ml; 18.8% susceptible) and meropenem (MIC50, 8 microg/ml; 50% susceptible) against Ralstonia pickettii. Since selection of the most appropriate antimicrobial agents for testing and reporting has not been established by the NCCLS for many of NF-GNB species, results from large multicenter studies may help to guide the best empiric therapy.

In vitro activity of tigecycline, a new glycylcycline, tested against 1,326 clinical bacterial strains isolated from Latin America

UNLABELLED The in vitro activity of tigecycline (former GAR-936), a new semisynthetic tetracycline, was evaluated in comparison with tetracycline and other antimicrobial agents. MATERIAL AND METHODS A total of 1,326 contemporary clinical isolates collected from the Latin American region were collected in 2000-2002 period and tested with microdilution broth according to the CLSI guidelines. The bacterial pathogens evaluated included Staphylococcus aureus (505), Streptococcus pneumoniae (269), coagulase-negative staphylococci (CoNS; 227), Haemophilus influenzae (129), Enterococcus spp. (80), Moraxella catarrhalis (54), beta-haemolytic streptococci (28), viridans group streptococci (26), and Neisseria meningitidis (8) RESULTS Tigecycline demonstrated excellent activity against all Gram-positive cocci, with 90% of penicillin-resistant S. pneumoniae strains being inhibited at 0.12 microg/mL, while the same isolates had an MIC90 of > 16 microg/mL for tetracycline. All Enterococcus spp. were inhibited at 0.25 microg/mL of tigecycline. Tigecycline (MIC50, 0.25 microg/mL) was eight-fold more potent than minocycline (MIC50, 2 microg/mL) against oxacillin-resistant S. aureus (ORSA); all ORSA were inhibited at < 2 microg/mL of tigecycline. Tigecycline demonstrated excellent activity (MIC50, 0.5 microg/mL) against CoNS with reduced susceptibility to teicoplanin (MIC, 16 microg/mL). Tigecycline also showed high potency against respiratory pathogens such as M. catarrhalis (MIC50, 0.12 microg/mL) and H. influenzae (MIC50, 0.5 microg/mL). No tigecycline resistant isolates were detected when the proposed susceptible breakpoints (< 4 microg/mL) was applied. CONCLUSIONS This results indicate that tigecycline has potent in vitro activity against clinically important pathogenic bacteria, including Gram-positive isolates resistant to both tetracycline and minocycline.

Molecular assessment of invasive Streptococcus pneumoniae serotype 1 in Brazil: evidence of clonal replacement

In Brazil, serotype 1 Streptococcus pneumoniae is one of the most prevalent causes of severe infection. This study investigated the genetic relatedness of 134 serotype 1 isolates obtained from invasive diseases during the period 1977-2005. Molecular typing by PFGE revealed two major lineages using visual inspection and computer analysis. Type A comprised 94 isolates (70.2 %) with four subtypes, whereas type B comprised 40 isolates (29.8 %) with eight subtypes. Subtype A3, the most frequent genotype, accounting for 65 % of the total isolates, was identified as a representative of clone Sweden(1)-40 (ST304). Type B was predominant in the period 1977-1988. In contrast, an increase in the type A lineage was detected from 1990 in Brazil, significantly associated with isolates recovered from pneumonia cases and from young patients. This study clearly established a temporal switch between two lineages of S. pneumoniae serotype 1 in Brazil, with a wide dispersion of clone Sweden(1)-40 in recent years.

Influence of Disk Preparation on Detection of Metallo-β-Lactamase-Producing Isolates by the Combined Disk Assay

ABSTRACT The combined disk assay has been used for detection of metallo-β-lactamase-producing isolates. We have observed that the size of inhibition zones produced by many β-lactam/metallo-β-lactamase inhibitor (IMBL) combinations may differ depending on the way that the combined disks were prepared. Among the 10 β-lactam/IMBL combinations tested, only the imipenem/EDTA combination produced similar results.

Evaluation of the Susceptibility profiles, genetic similarity and presence of qnr gene in Escherichia coli resistant to ciprofloxacin isolated in Brazilian hospitals

Increasing quinolone resistance has been reported worldwide, mainly among clinical isolates of Escherichia coli. The objectives of this study were to determine the susceptibility profile, the genetic relatedness, and the prevalence of the qnr gene among ciprofloxacin-resistant Escherichia coli isolated from distinct Brazilian hospitals. A total of 144 ciprofloxacin-resistant Escherichia coli were isolated from 17 Brazilian hospitals between January/2002 and June/2003. The antimicrobial susceptibility testing was performed by microdilution according to NCCLS. The presence of the qnr gene was initially screened by colony blotting, and then confirmed by PCR followed by DNA sequencing. Ninety-five urinary ciprofloxacin-resistant Escherichia coli were further selected for molecular typing by pulsed-field gel electrophoresis (PFGE). Imipenem and meropenem showed the highest susceptibility rates (100.0% for both compounds) followed by amikacin (91.0%) and piperacillin/tazobactan (84.8%). A single ciprofloxacin-resistant Escherichia coli isolate was positive for qnr among the 144 ciprofloxacin-resistant Escherichia coli. Forty-six PFGE patterns were observed among the 95 ciprofloxacin-resistant Escherichia coli type. This study shows that therapeutic options are limited for treatment of ciprofloxacin-resistant Escherichia coli due to the presence of additional mechanisms of antimicrobial resistance, such as ESBL production. The qnr gene was uncommon among ciprofloxacin-resistant Escherichia coli clinical isolates, but its identification might indicate the emergence of this mechanism of quinolone resistance in Brazil. The great genomic variability found among the ciprofloxacin-resistant Escherichia coli highlights the importance of the appropriate use of quinolone to restrict the selection of resistant isolates.

Progress towards meningitis prevention in the conjugate vaccines era

Acute bacterial meningitis is an important cause of morbidity and mortality among children less than five years old, Haemophilus influenzae, Streptococcus pneumoniae and Neisseria meningitidis being the most important agents of bacterial meningitis in developing countries. The development of the conjugate vaccines in the beginning of the 90s, especially type b H. influenzae (Hib), and more recently the heptavalent pneumococcal and the serogroup C meningococcal vaccines, have contributed directly to changes in the epidemiological profile of these invasive diseases (direct effect) and of their carriage status (indirect effect). We review the impact of the Hib conjugate vaccine in Latin American countries, where this vaccine has been implemented, and the potential of pneumococcal and meningococcal conjugate vaccines for the reduction of meningitis worldwide. We also address constraints for the development and delivery of these vaccines and review new candidate state-of-the-art vaccines. The greatest challenge, undoubtedly, is to implement these vaccines worldwide, especially in the developing regions.