Diagnostic Microbiology & Infectious Disease

Biofilm formation by Streptococcus pneumoniae strains and effects of human serum albumin, ibuprofen, N-acetyl-l-cysteine, amoxicillin, erythromycin, and levofloxacin

2010-08-01

Abstract: Streptococcus pneumoniae ability to produce biofilms may induce persistent infections and difficulties for eradication in vivo. We investigated the ability of 11 pneumococcal strains (serotypes 3, 6B, 9V, 19F, and 23F) to form biofilms on polystyrene plates at 16 and 24 h. The extent of biofilm was greater at 24 h in 10 strains, being the highest magnitude for serotype 3 strains. Human serum albumin at 25 000 μg/mL and ibuprofen at 128 μg/mL significantly reduced biofilm formation in 7 and 5 strains, respectively. Amoxicillin, erythromycin, and levofloxacin at supra-MIC concentrations were very active against planktonic cells of 3 selected strains but lower on biofilm-associated organisms in 2 strains and null against the third. Although N-acetyl-l-cysteine had very little activity against both planktonic and biofilm-forming organisms, when combined with the 3 antibiotics, a slightly enhanced activity against biofilm-embedded organisms in 1 strain and combined with amoxicillin in another one was observed.

Two-decade trends in primary Helicobacter pylori resistance to antibiotics in Bulgaria

2010-08-01

Abstract: Evaluating long-term trends in antibiotic resistance can predict earlier the short-term changes in resistance patterns. The aim of the present study was to compare primary resistance rates in 501 Helicobacter pylori strains in 2007 to 2009 to those in 1990 to 1995 (179 strains) and the antibiotic MICs to detect the 20-year resistance evolution. In 2007 to 2009, strains from children exhibited lower resistance rates to metronidazole (16.4%) and ciprofloxacin (2.7%) than those from adults (27.3% and 10.3%, respectively). In 2008 to 2009, more children (29.3%) harbored clarithromycin-resistant strains compared to the adults (17.4%). Overall clarithromycin resistance rate (19.4%) in 2007 to 2009 was much higher than that in 1990 to 1995 (6.2%). MIC90 of erythromycin in 1990 to 1995 was 142.2-fold lower than that of clarithromycin in 2007 to 2009. Clarithromycin MIC90 increased >42-fold since 2001 to 2004. Quinolone resistance rate increased 7.7-fold, being 9.2% in 2007 to 2009 versus 1.2% in 1990 to 1995, with a 5-fold increase in MIC90. Conversely, the amoxicillin resistance decreased from 3.2% in 1996 to 1999 to 0.4% in 2007 to 2009. The MIC90's of tetracycline remained stable but MIC50's of both metronidazole and tetracycline before 1996 decreased about 4-fold to 2007 to 2009. In conclusion, associations between the resistance evolution and patients' age groups as well as the national outpatient antibiotic use have been found. H. pylori resistance to antibiotics showed many long-term changes, with a more rapid evolution for clarithromycin than for the other antibiotics. Metronidazole and tetracycline did not show a resistance evolution but exhibited a decrease in MIC50 since 1990. The significant increase in ciprofloxacin resistance was found only by extending the study period to 20 years.

Dynamics of the laboratory results in patients with pulmonary tuberculosis

Stefan Panaiotov, Massimo Amicosante — 2010-08-01

Abstract: The process of infection and disease development is difficult to follow-up before tuberculosis (TB) confirmation. The laboratory analysis mirrors the infection with the possible subsequent breakdown to clinical TB. To better define the dynamics of the laboratory results in suspected and already confirmed patients with pulmonary TB, we studied the analysis of 1467 pathologic samples collected during the hospitalization of 841 patients. The samples were analyzed by 3 laboratory methods—direct microscopy, culture, and polymerase chain reaction (PCR). It was found that compared to cultures, the PCR method is more sensitive. For few cases, we demonstrate that the PCR result is positive about 2 weeks before the first positive culture. During the treatment follow-up, the PCR result remains positive for a long time, up to 4 to 5 months after the last positive culture. For better definition of the period during which microscopy and culture results remain positive, we studied the laboratory results of 100 casually selected patients with pulmonary TB positive on culture. The median periods during which these patients were found to be microscopy and culture positive were 10 and 25 days, respectively. Second to the dynamics of the laboratory results, we demonstrate that TB development is very rapid, whereas the period of recovery is long. The PCR results have to be reproducibly negative to accept that the process of active therapy is completed and the patient can remain under surveillance. On the basis of the laboratory data obtained, we propose empiric models for the dynamics of the laboratory results for patients with pulmonary tuberculosis.

Comparison of real-time polymerase chain reaction and conventional biochemical methods for identification of Mycobacterium chelonae–Mycobacterium abscessus group to the species level

Nora Guarin, Indre Budvytiene, Laleh Ghafghaichi, Niaz Banaei — 2010-08-01

Abstract: The Mycobacterium chelonae–Mycobacterium abscessus group (MCAG) is the most common cause of infections because of rapidly growing mycobacteria. Rapid identification of MCAG to the species level is essential for choosing empiric antibiotic treatment and for public health measures. In this study, we compared the performance of a single-tube multiplex, real-time polymerase chain reaction (PCR) assay to 3 biochemical tests for species-level identification of 46 MCAG isolates. We show that real-time PCR provides the most accurate results for rapid species-level identification of MCAG.

Identification and characterization of Schistosoma japonicum Sjp40, a potential antigen candidate for the early diagnosis of schistosomiasis

2010-08-01

Abstract: The pathogenesis of schistosomiasis is mainly caused by egg-induced granuloma formation and subsequent fibrosis. If Schistosoma japonicum infections could be detected in the early stage, especially before the egg deposition in the host tissues, the development of severe pathologic lesions might be prevented efficiently. The present study identified and characterized S. japonicum Sjp40, a potential antigen candidate for the early diagnosis of schistosomiasis. From the S. japonicum cercariae cDNA library, a clone encoding Sjp40 was identified by screening with the pooled rabbit sera collected on day 21 postinfection. Then, the recombinant Sjp40 protein (rSjp40) and monoclonal antibodies (McAbs) anti-rSjp40 were developed. The expression profiles of Sjp40 at 3 stages of S. japonicum, including egg, cercariae, and adult, were also determined at both mRNA and protein level, which displayed that the expression pattern of Sjp40 varied at different stages. Quantification of circulatory anti-Sjp40 IgG in the infected mice sera by time-resolved fluoroimmunoassay showed a statistically significant increase on days 21, 28, 35, and 42 postinfection compared with the mice sera prior to infection and the control mice. It was further confirmed by Western blot that all 8 clones of anti-rSjp40 McAbs could react specifically with the native antigen in S. japonicum cercariae, and rSjp40 could be recognized by the pooled infected mouse sera on days 21, 28, 35, and 42 postinfection as well as the pooled patient sera with acute schistosomiasis japonica. These findings indicated that Sjp40 and its antibodies are detectable from the host at a relatively early phase (day 21 postinfection with S. japonicum) and suggested that Sjp40 is a potential antigen candidate for the early diagnosis of schistosomiasis.

Detection of IgG-class antibodies to measles, mumps, rubella, and varicella-zoster virus using a multiplex bead immunoassay

2010-08-01

Abstract: Serologic testing for measles, mumps, rubella, and varicella (MMRV) IgG is traditionally performed by immunofluorescence assay or enzyme immunoassay (EIA). Although sensitive and specific, these methods are labor intensive, time consuming, and require separate assays for each analyte. This study evaluated the performance of the MMRV IgG AtheNA Multi-Lyte® assay using nonclinically characterized serum specimens submitted to our laboratory for routine MMRV IgG testing. Mumps (n = 492) or rubella (n = 500) IgG were initially tested by enzyme-linked fluorescent antibody (ELFA), whereas measles (n = 494) or varicella (n = 497) were analyzed by EIA. Each sample was also tested by the AtheNA Multi-Lyte assay. Discordant results were retested by the predicate method and the multiplex assay, with further discrepancies being arbitrated by a third test. Compared to EIA/ELFA for MMRV IgG, the AtheNA assay demonstrated an overall agreement of 97.4%, 98.2%, 97.6%, and 100%, respectively. Use of this multiplex assay allows for the simultaneous detection of MMRV IgG, potentially decreasing cost, sample volume requirements, aliquot errors, and hands-on testing time.

Trend of ciprofloxacin resistance in Neisseria gonorrhoeae strains isolated in Italy and analysis of the molecular determinants

2010-08-01

Abstract: A total of 599 Neisseria gonorrhoeae strains collected in Italy in 2 periods, 2003 to 2005 and 2007 to 2008, were screened for ciprofloxacin susceptibility by Etest. Ciprofloxacin-resistant strains (49.7%) were characterized by i) serovar determination, patterns of mutation in gyrA, and parC genes (38%, randomly selected) and ii) N. gonorrhoeae multiantigen sequence typing (56% of the strains isolated from patients who declared their sexual orientation). The percentage of ciprofloxacin-resistant strains increased from 42 (2003–2005) to 58 (2007–2008); in the second period, strains with MIC value >32 μg/mL have been observed. Mutations in gyrA and parC genes were identified in the majority of strains (88%). Ciprofloxacin-resistant isolates among men who have sex with men (MSM) increased from 24% in 2003 to 2005 to 47% in 2007 to 2008. However, sequence types exclusively found among MSM were mostly due to a single strain. This is the first study in Italy combining N. gonorrhoeae ciprofloxacin susceptibility testing with molecular analyses and comparing the results over time.

Characterization of macrolide resistance in Mycoplasma pneumoniae isolated from children in Shanghai, China

Yang Liu, Xinyu Ye, Hong Zhang, Xiaogang Xu, Wanhua Li, Demei Zhu, Minggui Wang — 2010-08-01

Abstract: One hundred Mycoplasma pneumoniae strains were isolated from pediatric patients from March 2008 to July 2009. Of 100 isolates, 90 (90%) were resistant to erythromycin (MICs >128 μg/mL for 88 strains and 64 μg/mL for 2 strains), azithromycin, and clarithromycin. Fluoroquinolones and tetracyclines maintain good activities against clinical M. pneumoniae isolates. Of 90 macrolide-resistant M. pneumoniae strains, 88 (98%) harbored an A-to-G transition mutation at position 2063 in 23S rRNA genes, and the remaining 2 showed either A2064G or A2063T mutation; the latter point mutation is newly discovered and reported. Ninety-three (93%) clinical isolates were classified into the P1 gene restriction fragment length polymorphism (RFLP) type I, and 7 (7%) were type II.

Worldwide summary of telavancin spectrum and potency against Gram-positive pathogens: 2007 to 2008 surveillance results

2010-08-01

Abstract: Telavancin was approved in the United States and Canada for the treatment of adult patients with complicated skin and skin-structure infections (cSSSI) caused by susceptible Gram-positive isolates. In this study, telavancin and comparator antimicrobial activities were determined against a total of 24 017 clinical isolates, including Staphylococcus aureus, coagulase-negative Staphylococcus spp. (CoNS), Enterococcus spp., and various Streptococcus spp. Overall, telavancin was highly active across all geographic regions for S. aureus (MIC50/90, 0.12/0.25 μg/mL; 100.0% susceptible), CoNS (MIC50/90, 0.12/0.25 μg/mL), vancomycin-susceptible Enterococcus faecalis (MIC50/90, 0.25/0.5 μg/mL; 100.0% susceptible), Enterococcus faecium (MIC50/90, 0.06/0.12 μg/mL), Streptococcus pneumoniae (MIC50/90, ≤0.015/0.03 μg/mL), viridans group Streptococcus spp. (MIC50/90, 0.03/0.06 μg/mL; 100.0% susceptible), and β-hemolytic Streptococcus spp. (MIC50/90, 0.03/0.12 μg/mL; 99.8% susceptible). Telavancin had potent activity against vancomycin-nonsusceptible, teicoplanin-susceptible (VanB) E. faecalis (MIC50/90, 0.25/0.5 μg/mL) and E. faecium (MIC50/90, 0.06/0.25 μg/mL). These in vitro results show continued activity for telavancin, which represents an important alternative available for treating cSSSI.

Macrolide-resistant Streptococcus pyogenes from Chinese pediatric patients in association with Tn916 transposons family over a 16-year period

2010-08-01

Abstract: To investigate changes in the antimicrobial susceptibility of Streptococcus pyogenes isolates over a 16-year period, 456 group A streptococci isolates were collected from Chinese pediatric patients among 1993 to 1994 and 2005 to 2008. Susceptibilities to antibiotics were performed using agar dilution methods. The macrolide resistance genes ermB, ermTR, mefA, and tetracycline-resistant gene tetM and the int and xis genes of Tn916 family were detected by polymerase chain reaction. All 456 strains were analyzed by emm typing. Selected strains representing each emm type were further characterized by pulsed-field gel electrophoresis. The resistance rates of erythromycin and clindamycin both significantly increased during the 2 sample periods (79.7% versus 94% for erythromycin and 75.4% versus 96.9% for clindamycin). Telithromycin resistance rate increased from 20.37% to 87.93%. Among the macrolide resistance strains, the rate of strains with the genes int, xis, tetM, and ermB increased with time (16.05% versus 86.91%, P < 0.05). The emm1 and emm12 isolates had high rates of ermB gene, which increased after 16 years (65.2% versus 86.23% for emm1 and 7.7% versus 91.8% for emm12). This study demonstrates the increase in macrolide resistance in S. pyogenes in Chinese children over a 16-year period. The phenomenon may be related not only with the shift in the emm types but also with the change of macrolide-resistant mechanisms. The change of Tn916 family among the isolates may be related with the increased resistance.

Assessment of prevalence and changing epidemiology of extended-spectrum β-lactamase–producing Enterobacteriaceae fecal carriers using a chromogenic medium

2010-08-01

Abstract: Five hundred fecal samples from 462 patients (68.4% ambulatory) (February–April, 2007) from Madrid (Spain) were screened for extended-spectrum β-lactamase (ESBL) producers using ceftazidime and cefotaxime (1 mg/L) MacConkey (MAC) agar plates and a chromogenic media (chromID ESBL; bioMérieux, Marcy-l'Etoile, France). blaESBL, qnr, aac(6′)Ib-cr, and 16S rRNA methylase genes were assessed. A prevalence of 8.2% of ESBL fecal carriers was observed (8.9% hospitalized, 7.9% nonhospitalized patients), higher than that previously observed (1991, 0.6%; 2003, 7.0%). Sensitivity, specificity, and positive and negative predicted values were 100%, 94.8%, 63%, and 100% for chromID ESBL and 87.8%, 89.8%, 43.4%, and 98.9% for MAC, respectively. ESBL distribution was as follows: CTX-M-9-group, 40% (mainly CTX-M-14); CTX-M-1-group, 26.6% (mainly CTX-M-15); SHV-type, 29% (mainly SHV-12); and TEM-type, 4.4%. These enzymes were found in pulsed-field gel electrophoresis nonclonally related Escherichia coli and Klebsiella pneumoniae isolates. Transferable quinolone resistance was confirmed in CTX-M-9 (qnrS1), CTX-M-15 [aac(6′)Ib-cr, qnrS1], and SHV-12 (qnrB7, qnrS1) producers but not 16S rRNA methylase genes. The chromID ESBL medium was reliable to screen ESBL fecal carriers with a general decrease in the laboratory workload. Time-to-time monitoring of ESBL fecal carriers is useful to ascertain current trend of ESBL epidemiology.

High percentage of resistance to ciprofloxacin and qnrB19 gene identified in urinary isolates of extended-spectrum β-lactamase–producing Escherichia coli in Madrid, Spain

2010-08-01

Abstract: The presence of qnr genes in 191 extended-spectrum β-lactamase–producing Escherichia coli from 2005, with 75% of resistance to ciprofloxacin, was evaluated. An SHV-12–producing E. coli carried qnrB19; both genes were transferred by conjugation. No qnrA- or qnrS-positive strains were detected. In addition, we identified 3 new parC mutations (S80W, E84R, and E84A).

Evaluation of Vitek2 and BD Phoenix in antimicrobial susceptibility testing of Acinetobacter baumannii and Pseudomonas aeruginosa

2010-08-01

Abstract: The accuracy of antimicrobial susceptibility testing of Vitek2 and BD Phoenix against Acinetobacter baumannii and Pseudomonas aeruginosa was evaluated. Both systems showed overall categoric agreement of ≤90% for cefepime and ceftazidime against A. baumannii and imipenem and cefepime (and ceftazidime with Vitek2) against P. aeruginosa because of high minor error rates.

Comparison of CMY-2 plasmids isolated from human, animal, and environmental Escherichia coli and Salmonella spp. from Canada

2010-08-01

Abstract: A total of 244 CMY-2 plasmids from 5 separate studies involving Escherichia coli and Salmonella human clinical cases as well as E. coli from feedlots and water sources were examined. Genetically similar CMY-2 plasmids isolated from either E. coli or Salmonella from human, animal, and environmental sources are widely distributed across Canada and cluster into replicon types I1, A/C, and K/B and an unidentified group.

Aspergillus DNA contamination in blood collection tubes

2010-08-01

Abstract: Fungal polymerase chain reaction (PCR)-based diagnostic methods are at risk for contamination. Sample collection containers were investigated for fungal DNA contamination using real-time PCR assays. Up to 18% of blood collection tubes were contaminated with fungal DNA, probably Aspergillus fumigatus. Lower proportions of contamination in other vessels were observed.

Methicillin-resistant Staphylococcus aureus resistance to non–β-lactam antimicrobials in the United States from 1996 to 2008

2010-08-01

Abstract: We report the resistance rates of Staphylococcus aureus to non–β-lactam antimicrobials from The Surveillance Network Database-USA (Eurofins-Medinet, Chantilly, VA). Specimens studied were from lower respiratory tract, wounds, and blood. Patients were stratified by age group and patient setting. There were 2,053,219 isolates of S. aureus and 973,116 of methicillin-resistant S. aureus (MRSA). The MRSA rate increased until 2004 and then leveled off. MRSA showed decreasing resistance to tetracycline and trimethoprim–sulfamethoxazole (TMP–SMX). By age group, the greatest MRSA rate increase was for individuals 17 years and younger. Non–β-lactam antimicrobials and particularly TMP–SMX should be considered therapeutic options for staphylococcal infections.

Rapid detection of H and R Panton–Valentine leukocidin isoforms in Staphylococcus aureus by high-resolution melting analysis

2010-08-01

Abstract: We designed a single-step, closed-tube, real-time polymerase chain reaction and high-resolution melting assay to simultaneously detect the presence of the Panton–Valentine leukocidin gene and discriminate histidine and arginine isoforms. Of 223 Staphylococcus aureus isolates from northern Australia, isoforms clustered by clonal complex (CC). All CC93 isolates harbored the arginine isoform.

Comparison of the analytic sensitivities of a recombinant immunoblot assay and the radioimmune precipitation assay for the detection of antibodies to Trypanosoma cruzi in patients with Chagas disease

2010-08-01

Abstract: The diagnosis of chronic Chagas disease usually is made by detecting antibodies to Trypanosoma cruzi, the protozoan parasite that causes this illness. A highly sensitive and specific immunoblot assay developed by us showed a higher analytic sensitivity than the radioimmune precipitation assay, which is used widely as a confirmatory test.

Erratum to “Disseminated Rhodococcus equi infection in a kidney transplant patient without initial pulmonary involvement” [Diagn Microbiol Infect Dis 2009 Dec;65(4):427–30]

2010-08-01

On behalf of the authors, Dr. J.R. Lo-Ten-Foe states that they were not able to adequately reproduce the identification of the causative bacterial strain in the particular patient. Therefore, they cannot be certain about the correct identification.