Direct identification of major Gram-negative pathogens in respiratory specimens by respiFISH® HAP Gram (-) Panel, a beacon-based FISH methodology.

Rapid detection of microorganisms in respiratory specimens is of paramount importance to drive the proper antibiotic regimen to prevent complications and transmission of infections. In the present study, the respiFISH® HAP Gram (-) Panel (miacom diagnostics GmbH, Duesseldorf, Germany) for the etiological diagnosis of hospital-acquired pneumonia was compared with the traditional culture method for the detection of major Gram-negative pathogens in respiratory specimens. respiFISH® combined the classical fluorescence in situ hybridization (FISH) technology with fluorescence-labeled DNA molecular beacons as probes. From September 2011 to January 2012, 165 samples were analyzed: the sensitivity and specificity were 94.39 and 87.93%, respectively. Only six pathogens (3.6%) were not identified with respiFISH®, while seven specimens (3%) provided false-positive results. This beacon-based identification shortens the time to result by at least one work day, providing species-level identification within half an hour. Considering the high sensitivity and specificity and the significant time saving, the introduction of bbFISH® assays could effectively complement traditional systems in microbiology laboratories.

Interference of confounding factors on the use of (1,3)-beta-D-glucan in the diagnosis of invasive candidiasis in the intensive care unit.

Invasive fungal infections (IFIs) are an increasing problem in intensive care units (ICUs), and conventional diagnostic methods are not always reliable or timely enough to deliver appropriate antimicrobial therapy. The dosage of fungal antigens in serum is a promising diagnostic technique, but several confounding factors, such as treatment with immunoglobulins (Ig), albumin, or antifungals, could interfere with the correct interpretation of the (1,3)-beta-D-glucan (BG) assay. This study assessed the reliability of the BG assay and the influence of timing and dosage of major confounding factors on circulating levels of IFI biomarkers. 267 ICU patients who underwent a BG assay were retrospectively studied. The timing and dosage of albumin, use of azole treatment, and infusions of intravenous IgG, red blood cells, concentrated platelets, and frozen plasma were analyzed to find possible correlations with the BG results. The sensitivity and specificity of the BG assay were calculated. The BG test in serum showed high sensitivity (82.9 %) but low specificity (56.7 %). The optimal cut-off for the test was 95.9 pg/mL. The mean BG level in proven invasive candidiasis was around 400 pg/mL. The only factor that was found to significantly confound (p < 0.05) the diagnostic performance of the BG assay was the administration of more than 30 g of albumin within 2 days prior to BG testing. The BG assay remains a useful diagnostic test in ICU patients and the levels of BG are useful in evaluating the positive predictive value of this biomarker. The only confounding factor in our study was the use of albumin.

Intrinsic penicillin resistance in enterococci.

Penicillin resistance development in enterococci has been associated with overproduction of a low-affinity penicillin-binding protein (PBP) that is a normal component of the PBP pattern of these bacteria and is apparently able to substitute the functions of the other PBPs. In resistant mutants of Enterococcus hirae ATCC 9790 the low-affinity PBP (PBP5) overproduction was associated with a deletion in a genetic element, located 1 kb upstream of the pbp5 gene, which negatively controlled PBP5 synthesis. Hypersusceptibility to penicillin was associated with a point mutation in the pbp5 gene, which causes premature termination of translation. Structural homologies between low-affinity PBPs of the different enterococcal species have been suggested by cross-reactivity of antibodies raised against E. hirae PBP5 with PBP5 of Enterococcus faecium and Enterococcus faecalis. Acquisition of a high-level ampicillin resistance in E. faecium was associated with overproduction of PBP5, which, compared with PBP5 of moderately resistant strains, appeared to be modified in its penicillin-binding capability. The modified phenotype of PBP5 was found to be associated to some amino acid substitutions in the region between the SDN and KTG motifs. In particular, the substitution converting a polar residue (T) in a nonpolar one (A or I) could play an important role in remodeling the penicillin-binding domain and determining the decrease in penicillin affinity.

Molecular analysis of vanA enterococci isolated from humans and animals in northeastern Italy.

A total of 53 vancomycin-resistant vanA-positive enterococci isolates from poultry farms (17 Enterococcus faecium; 8 Enterococcus durans) and from different hospitals (23 E. faecium; 5 Enterococcus faecalis) in northeastern Italy were compared on the basis of their antibiotic susceptibilities, their SmaI pulsed-field gel electrophoresis (PFGE) patterns, and the organization of their Tn1546-related elements. Ampicillin resistance was similar in both groups of isolates (52 and 60.7%, respectively), whereas human strains were more resistant to high-level gentamicin and streptomycin. A total of 52% of animal strains and 60% of human strains were resistant to tetracycline, and 56% and 46.4% to quinupristin/dalfopristin, respectively. In E. faecium and E. durans animal isolates, nine and six distinct PFGE patterns, respectively, were found: in two instances indistinguishable isolates were found from different farms. In E. faecium and E. faecalis human isolates, nine and six distinct PFGE patterns, respectively, were found; among E. faecium strains, 12 were identical or closely related and were isolates from the same hospital. Elements mediating vanA-glycopeptide resistance were characterized by PCR with primers that amplified 10 overlapping fragments of Tn1546. A total of 84.6% of animal strains and 64.2% of human strains contained elements indistinguishable from the prototype Tn1546. In addition, nine different types were identified, but none was common to animal and human strains.

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.

Development of a rapid method for quantitative evaluation of Mycobacterium tuberculosis growth based on competitive polymerase chain reaction.

A competitive polymerase chain reaction (cPCR) assay for the quantitative evaluation of Mycobacterium tuberculosis growth was developed based on co-amplification of genomic DNA and a modified DNA fragment derived from a well-conserved region of the 16S rRNA gene. There was a good correlation between the number of DNA copies in the sample, indicated by competitive PCR, and the number of colony forming units determined by conventional culture methods.

In vitro activity of lomefloxacin (SC-47111) against enterobacteriaceae, enterococci and staphylococci.

The activity of lomefloxacin, a new difluorinated quinolone, was tested against 190 Enterobacteriaceae strains (belonging to 23 different species), 70 enterococci and 70 staphylococci. As regards Enterobacteriaceae, the activity of lomefloxacin was the same as that of norfloxacin in 9 out of the 23 species tested, and only slightly lower in further 8 species. Minimum inhibitory concentrations (MIC) values for 90% of strains were 0.5 microgram/ml in 2 species, 0.25 microgram/ml in 6, 0.125 microgram/ml in 4, and lower than 0.125 microgram/ml in 8. Slightly higher values were obtained for Serratia marcescens (2 micrograms/ml), whilst, as already reported for the other new quinolones, the susceptibility of the Providencia genus was very poor, with MIC values up to 128 micrograms/ml for the vast majority of strains. Lomefloxacin proved bactericidal at the MIC in all the Enterobacteriaceae strains tested but 20. In the latter strains, however, bactericidal activity could be appreciated at values slightly exceeding MIC. As regards enterococci, the MIC for 90% of strains was 32 micrograms/ml. Minimum bactericidal concentration (MBC) was the same as the MIC for 78% of the strains tested and was only twofold higher in all the others. The new drug was also active against staphylococci having an MIC50 and MIC90 of 0.5 and 2 micrograms/ml, respectively. It was bactericidal at the MIC for 62% of the strains and at twofold the MIC for all the others.

In vitro activity of cefpirome (HR 810) against enterococci and staphylococci.

The inhibitory activity of cefpirome (HR 810), a new cephalosporin derivative for parenteral use, was tested by agar dilution methods against Enterococcus faecalis (100 strains), Staphylococcus aureus (40 strains) and coagulase-negative staphylococcal species (60 strains) in comparison with other beta-lactam antibiotics. For E. faecalis, the cefpirome minimum inhibitory concentration (MIC) range was 2-128 micrograms/ml, with an MIC50 of 8 micrograms/ml, and an MIC90 of 64 micrograms/ml. The optimal bactericidal activity against strains with MICs of < or = 8 micrograms/ml occurred at 2-4 times the MIC, and the reduction in the initial inoculum was 99.9-99.7% after 24 h incubation at these concentrations. Mec gene-negative staphylococci (both S. aureus and coagulase-negative species) had cefpirome MICs of 0.25-2 micrograms/ml (MIC50 0.5 microgram/ml, MIC90 1 microgram/ml). Mec gene-positive strains had MICs of 0.5-128 micrograms/ml (MIC50 2 micrograms/ml, MIC90 32 micrograms/ml). Strains with borderline resistance to oxacillin which did not harbor the mec gene and which were susceptible to cefpirome maintained their susceptibility even when high-density inocula were used and after several passages in media containing the antibiotic. These studies present some potential advantages of cefpirome over other cephalosporins in the inhibitory activity against Gram-positive cocci.

PBP binding and periplasmic concentration as determinants of the antibacterial activities of three new oral cephalosporins in Escherichia coli.

The antibacterial activities of cefetamet, cefixime and cefuroxime were investigated in Escherichia coli with regard to their penetration rates through the outer membrane and their affinities for PBPs in Escherichia coli. The permeability coefficient of cefetamet was measured in E. coli C600 carrying a pUC18 plasmid derivative, in which the gene of a transferable cephamycinase (CMY-2) was cloned, whereas diffusion of cefixime and cefuroxime was measured in E. coli C600 harbouring the OXA-1 beta-lactamase gene. It was found that cefetamet penetrated 5 and 9 times faster than cefixime and cefuroxime, respectively. The correlation between antibacterial activities and PBP affinities was studied in E. coli C600 not producing beta-lactamases. In this strain, cefetamet and cefixime shared the same inhibitory activity (MIC = 1 microgram/ml for both antibiotics) and the same affinity for PBP 3 (ID50 = 0.25 micrograms/ml). Cefuroxime had a lower inhibitory activity (MIC = 4 micrograms/ml) and a lower affinity for PBP 3 (ID50 = 0.5 microgram/ml). Cefixime and cefuroxime had 20 and 10 times higher affinity, respectively, than cefetamet for PBP 1s. It was concluded that the superior PBP affinity of cefixime can counterbalance the better penetration of cefetamet through the outer membrane, whilst differences in either permeability or PBP affinity seem sufficient to explain the lower antibacterial activity of cefuroxime compared to cefixime, which might well be related to the poor stability of the cefuroxime molecule.

A case of Beauveria bassiana keratitis confirmed by internal transcribed spacer and LSU rDNA D1-D2 sequencing.

We describe a case of fungal keratitis due to Beauveria bassiana in a farmer with Fuchs' dystrophy, treated with amphotericin B. Surgery with penetrating keratoplasty was necessary to resolve the lesions. Susceptibility testing and molecular sequencing permitted the identification and treatment of this rare aetiological agent of invasive fungal disease.

Rapid molecular technique analysis of a KPC-3-producing Klebsiella pneumoniae outbreak in an Italian surgery unit.

The rapid emergence of KPC-producing Klebsiella pneumoniae has become a serious problem in health-care settings, increasing in frequency worldwide. These infections are worrisome, since the antimicrobial treatment options for infections due to multidrug-resistant strains are very limited, and outbreaks must be rapidly detected and controlled. A semi-automated, repetitive-sequence-based PCR (rep-PCR) instrument (DiversiLab system) was evaluated in comparison with the pulse-field gel electrophoresis (PFGE) and multilocus sequence typing to investigate the outbreak of KPC-producing K. pneumoniae in a surgery unit at the University Hospital of Verona, Italy, as a rapid method for outbreak investigations. A selection of seven epidemiologically related K. pneumoniae showing resistance to carbapenem and three epidemiologically unrelated K. pneumoniae isolates were collected from patient with hospital-acquired infection. Among the epidemiologically related isolates, PFGE and Rep-PCR identified a unique pattern with more than 90% of homology. The concordance between DiversiLab and PFGE results confirmed the usefulness of rapid molecular techniques to investigate outbreaks due to multidrug-resistant bacteria. Moreover, this result could meet the international need for a harmonised typing tool, allowing the implementation of strict control measures to prevent dissemination of these organisms in health-care settings.

First case of bloodstream infection due to Candida magnoliae in a Chinese oncological patient.

We report a case of fungemia caused by Candida magnoliae, a yeast never associated with human disease. The infection occurred in a 42-year-old Chinese patient with gastric cancer complicated by peritoneal carcinosis. Multiple blood cultures were positive for yeast; the species was well identified with biochemical and molecular methods. The phylogenetic analysis showed a close relationship of C. magnoliae to Candida krusei.

Detection and quantification of hepatitis B virus DNA by SYBR green real-time polymerase chain reaction.

A single-round real-time polymerase chain reaction (PCR) assay based on SYBR green dye technology for the detection and quantification of hepatitis B virus (HBV) DNA in serum was evaluated and compared with a qualitative nested PCR and the Cobas Amplicor HBV Monitor assay (Roche Molecular Diagnostics, Milan, Italy). The performance of the real-time PCR assay was evaluated in a routine clinical laboratory setting with a total of 212 clinical specimens. The sensitivity of the real-time PCR corresponded to 31 IU/ml (70 copies/ml), and comparison with the qualitative nested PCR showed significant concordance for 94% of samples. The linear curve over 7 log units, spanning 10(3)-10(9) IU/ml (2.28 x 10(3) to 2.28 x 10(9) copies/ml), was observed in the quantitative determination. The interexperimental variability coefficient of the assay ranged from 0.22 to 0.39 and the intraexperimental variability coefficient from 0.24 to 0.41. By excluding values outside of the dynamic ranges of both tests, the HBV Monitor and the real-time PCR gave an agreement within +/-1 log unit for 90% of samples, while those for the remaining 10% were found to be above 1 log unit but less than 1.5 log units. When the results inside and outside the dynamic range of the HBV Monitor were examined, 90% of the results were in agreement. In conclusion, the real-time PCR based on SYBR green technology proved suitable for routine diagnostic purposes, showing good sensitivity, high specificity, high reproducibility, and good linearity over a broad dynamic range of quantification.

Identification of a genetic element (psr) which negatively controls expression of Enterococcus hirae penicillin-binding protein 5.

Enterococcus hirae ATCC 9790 produces a penicillin-binding protein (PBP5) of low penicillin affinity which under certain conditions can take over the functions of all the other PBPs. The 7.1-kb EcoRI fragment containing the pbp5 gene of this strain and of two mutants, of which one (E. hirae R40) overproduces PBP5 and the other (E. hirae Rev14) does not produce PBP5, was cloned in pUC18 and sequenced. In the 7.1-kb EcoRI fragment cloned from strain ATCC 9790, an open reading frame (psr) potentially encoding a 19-kDa protein was identified 1 kb upstream of the pbp5 gene. An 87-bp deletion in this element was found in the 7.1-kb EcoRI fragment cloned from strains R40 and Rev14. In addition, several base substitutions were found in the pbp5 genes of strains R40 and Rev14. One of these converted the 42nd codon, TCA, to the stop codon, TAA, in the pbp5 gene of Rev14. Escherichia coli strains were transformed with plasmids carrying the 7.1-kb EcoRI insert or a 2.6-kb HincII insert containing only the pbp5 gene of the three strains. Immunoblotting analysis of proteins expressed by these transformants showed that the 87-bp deletion in psr was associated with the PBP5 overproducer phenotype of strain R40 and the conversion of the TCA codon to the stop codon was associated with the PBP5 nonproducer phenotype of strain Rev14. None of the other nucleotide substitutions had any apparent effect on the level of PBP5 synthesized.

Modification of penicillin-binding protein 5 associated with high-level ampicillin resistance in Enterococcus faecium.

High-level ampicillin resistance in Enterococcus faecium has been shown to be associated with the synthesis of a modified penicillin-binding protein 5 (PBP 5) which had apparently lost its penicillin-binding capability (R. Fontana, M. Aldegheri, M. Ligozzi, H. Lopez, A. Sucari, and G. Satta. Antimicrob. Agents Chemother. 38:1980-1983, 1994). The pbp5 gene of the highly resistant strain E. faecium 9439 was cloned and sequenced. The deduced amino acid sequence showed 77 and 54% homologies with the PBPs 5 of Enterococcus hirae and Enterococcus faecalis, respectively. A gene fragment coding for the C-terminal part of PBP 5 containing the penicillin-binding domain was also cloned from several E. faecium strains with different levels of ampicillin resistance. Sequence comparison revealed a few point mutations, some of which resulted in amino acid substitutions between SDN and KTG motifs in PBPs 5 of highly resistant strains. One of these converted a polar residue (the T residue at position 562 or 574) of PBP 5 produced by susceptible and moderately resistant strains into a nonpolar one (A or I). This alteration could be responsible for the altered phenotype of PBP 5 in highly resistant strains.

Utility of molecular identification in opportunistic mycotic infections: a case of cutaneous Alternaria infectoria infection in a cardiac transplant recipient.

We report on a case of cutaneous infection caused by Alternaria infectoria in a cardiac transplant recipient. A rapid molecular diagnosis was obtained by sequence analysis of the internal transcribed spacer domain of the 5.8S ribosomal DNA region amplified from colonies developed on Sabouraud medium. Treatment consisted of a combination of systemic antifungal therapy, first with amphotericin B and then with itraconazole.

In vitro response to bactericidal activity of cell wall-active antibiotics does not support the general opinion that enterococci are naturally tolerant to these antibiotics.

The incidence of tolerance and paradoxical response to bactericidal activity of penicillin was investigated in 50 clinical isolates of Enterococcus faecalis. Of the isolates tested, 86% exhibited the paradoxical phenomenon whereby there were more survivors at high than at low concentrations above the MIC. Low penicillin concentrations caused decreases equal to or higher than 99.9% in 11 strains, from 99.9 to 99.5% in 23 strains, and lower than 99.5% in 9 strains. Of the total strains, 14% were killed to the same extent by all concentrations above the MIC. The bactericidal activities of other beta-lactams (ampicillin and piperacillin) and other cell wall inhibitors (vancomycin and daptomycin) were also tested against some of these strains. In general, beta-lactams exhibited the best bactericidal activity at 2 x MIC. Piperacillin was the most active, as at 2 x MIC it reduced the original inoculum by 99.9% or more in most of the strains. No concentration of vancomycin above the MIC caused 99.9% killing of the strains, whereas daptomycin was bactericidal at 8 x MIC in most cases. Paradoxical response to bactericidal activity of beta-lactams was abolished by incubation of the inoculum with 2 x MIC before exposure to higher antibiotic concentrations. These findings suggest that enterococci are not always tolerant to cell wall-active antibiotics and that accurate in vitro bactericidal tests may be useful for the choice of appropriate therapy for infections caused by these microorganisms.

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.