Marco Ligozzi

Identification of 54 mycobacterial species by PCR-restriction fragment length polymorphism analysis of the hsp65 gene

ABSTRACT A total of 121 reference and clinical strains of both slowly and rapidly growing mycobacteria belonging to 54 species were studied for restriction fragment length polymorphism of a PCR-amplified 439-bp segment of the gene encoding the 65-kDa heat shock protein. Restriction digests were separated by 10% polyacrylamide gel electrophoresis (PAGE). By including a size standard in each sample, the restriction fragment profile was calculated using a computer-aided comparison program. An algorithm describing these 54 species (including 22 species not previously described) is proposed. We found that this assay based on 10% PAGE provided a more precise estimate than that based on agarose gel electrophoresis of the real size of restriction fragments as deduced from the sequence analysis and allowed identification of mycobacteria whose PCR-restriction fragment length polymorphism analysis patterns were unequivocally identified by fragments shorter than 60 bp.

Evaluation of the VITEK 2 System for Identification and Antimicrobial Susceptibility Testing of Medically Relevant Gram-Positive Cocci

ABSTRACT A study was conducted to evaluate the new VITEK 2 system (bioMérieux) for identification and antibiotic susceptibility testing of gram-positive cocci. Clinical isolates of Staphylococcus aureus (n = 100), coagulase-negative staphylococci (CNS) (n = 100), Enterococcus spp. (n = 89), Streptococcus agalactiae (n = 29), and Streptococcus pneumoniae (n = 66) were examined with the ID-GPC identification card and with the AST-P515 (for staphylococci), AST-P516 (for enterococci and S. agalactiae) and AST-P506 (for pneumococci) susceptibility cards. The identification comparison methods were the API Staph for staphylococci and the API 20 Strep for streptococci and enterococci; for antimicrobial susceptibility testing, the agar dilution method according to the procedure of the National Committee for Clinical Laboratory Standards (NCCLS) was used. The VITEK 2 system correctly identified to the species level (only one choice or after simple supplementary tests) 99% of S. aureus, 96.5% of S. agalactiae, 96.9% of S. pneumoniae, 92.7% of Enterococcus faecalis, 91.3% of Staphylococcus haemolyticus, and 88% of Staphylococcus epidermidis but was least able to identify Enterococcus faecium (71.4% correct). More than 90% of gram-positive cocci were identified within 3 h. According to the NCCLS breakpoints, antimicrobial susceptibility testing with the VITEK 2 system gave 96% correct category agreement, 0.82% very major errors, 0.17% major errors, and 2.7% minor errors. Antimicrobial susceptibility testing showed category agreement from 94 to 100% for S. aureus, from 90 to 100% for CNS, from 91 to 100% for enterococci, from 96 to 100% for S. agalactiae, and from 91 to 100% for S. pneumoniae. Microorganism-antibiotic combinations that gave very major errors were CNS-erythromycin, CNS-oxacillin, enterococci-teicoplanin, and enterococci-high-concentration gentamicin. Major errors were observed for CNS-oxacillin and S. agalactiae-tetracycline combinations. In conclusion the results of this study indicate that the VITEK 2 system represents an accurate and acceptable means for performing identification and antibiotic susceptibility tests with medically relevant gram-positive cocci.

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. Images

Resistance of Streptococcus pyogenes to Erythromycin and Related Antibiotics in Italy

A survey of antimicrobial resistance in Streptococcus pyogenes, performed within the framework of a national surveillance program, has revealed a dramatic increase in resistance of S. pyogenes to erythromycin in most areas of Italy. In virtually all the centers that provided data for 3 consecutive years, the incidence of erythromycin-resistant strains increased twofold to 20-fold from 1993 to 1995 and was greater than 30% in five of the 14 centers participating in the study. The clonality of erythromycin-resistant isolates was studied in 15 strains isolated from different patients at the Institute of Microbiology of Verona University (Verona). The features of the Verona isolates and the substantially different rates of erythromycin and clindamycin resistance observed in most centers suggest that the spread of different resistance genes in multiple clones might be occurring throughout the country.

Overproduction of a low-affinity penicillin-binding protein and high-level ampicillin resistance in Enterococcus faecium

Five ampicillin-resistant clinical isolates of Enterococcus faecium were analyzed for a correlation between overproduction of the low-affinity penicillin-binding protein (PBP 5) and the level of ampicillin resistance. Comparison was made with one susceptible clinical isolate and its ampicillin-resistant derivative obtained in the laboratory by selection with increasing concentrations of penicillin. Overproduction of the low-affinity PBP relative to the susceptible isolate was noted in moderately resistant strains (MIC, 32 micrograms/ml) but not in highly resistant strains (MIC, 128 micrograms/ml). Polyclonal antibodies specifically reacting with the low-affinity PBP of Enterococcus hirae, Enterococcus faecalis, and Enterococcus faecium (M. Ligozzi, M. Aldegheri, S. C. Predari, and R. Fontana, FEMS Microbiol. Lett. 83:335-340, 1991) were used to determine the amount of this PBP in the E. faecium isolates. In all strains, the antibody preparation reacted with a membrane protein of the same molecular mass as PBP 5. The amount of this protein was very small in the susceptible strain but large in all of the resistant strains. These results suggest that the highly resistant strains also overproduced the low-affinity PBP, which, compared with PBP 5 of moderately resistant strains, appeared to be modified in its penicillin-binding capability. Images

Vancomycin-Resistant Enterococcus faecium Isolates Causing Hospital Outbreaks in Northern Italy Belong to the Multilocus Sequence Typing C1 Lineage

Multilocus sequence typing (MLST) was used to obtain insights into the genetic relationships between 14 vancomycin-resistant Enterococcus faecium (VREF) isolates from humans (hospitalized patients, 5 strains) and nonhuman sources (meat and poultry, 9 strains) in northern Italy over the period 1993-2001. The typing scheme (Homan et al., 2002, J. Clin. Microb., 40:1963-1971) based on seven housekeeping genes--adk (adenylate kinase), atpA (ATP synthase, alpha subunit), ddl (D-alanine-D-alanine ligase), gyd (glyceraldehyde-3-phosphate dehydrogenase), gdh (glucose-6-phosphate dehydrogenase), purK (phosphoribosylaminoimidazole carboxylase ATPase subunit), and pstS (phosphate ATP-binding cassette transporter)--was used. In the 14 VREF analyzed, the number of unique alleles ranged from 1 (gyd) to 8 (atpA). Isolates from hospitalized patients were defined by the unique allele purK 1. Nine sequence types (STs) were identified. All of the epidemic strains isolated over the period 2000-2001 showed identical or closely related pulsed-field gel electrophoresis (PFGE) patterns and clustered in the same ST78. These strains shared six of the seven alleles with the strain CA20 representative of the 1993-1999 outbreaks, which PFGE indicated as being unrelated to those of the recent outbreaks. MLST confirmed the unrelatedness of human and nonhuman strains already detected by PFGE. All isolates clustered in three main genetic lineages: group A comprised two of the three isolates from meat; group C the human strains of all outbreaks and one poultry strain; and group B four of the five poultry strains and one meat strain. All human strains carried the esp gene and clustered in the C1 sublineage that has been described as having emerged recently worldwide.

Vancomycin-resistantBacillus circulans carrying thevanA gene responsible for vancomycin resistance in enterococci

Acquired resistance to glycopeptide antibiotics was first described in enterococci, primarily Enterococcus faecium and Enterococcus faecalis (1). The potential spread of vancomycin-resistance determinants from enterococci to other gram-positive organisms is a serious public health concern. Most vancomycin-resistant enterococci possess the inducible high-level type of resistance encoded by the vanA gene cluster, which is carried on transposons similar or related to Tn1546 (2).

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

ABSTRACT 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.

Emergence of linezolid resistance in Enterococcus faecium not dependent on linezolid treatment

Despite their apparent lack of virulence, the enterococci have emerged as important nosocomial pathogens in recent years. Their intrinsic resistance against many antibiotics and their acquisition of resistance to vancomycin has played a prominent role in rapidly decreasing the therapeutic options available for infections caused by these organisms. Linezolid, a synthetic oxazolidinone antibiotic, was first introduced as a new therapeutic option against gram-positive cocci in early 2000. Following the start of its clinical use, resistance began to emerge, albeit at a very low rate, in clinical isolates of enterococci, and this resistance has been found to be dependent on prior linezolid exposure and duration of therapy [1]. We report here the isolation of a linezolid-resistant vancomycin-resistant E. faecium (LRVRE) strain from a patient who had never been treated with linezolid and who lacked risk factors known to be associated with the development of infections with vancomycin-resistant enterococci (VRE). The patient was a 64-year-old female who was admitted to the intensive cure unit (ICU) of our hospital in October 2004 after suffering a stroke and who died 8 days after admission. Prior to this admission, she had lived in her own home, had not been hospitalized during the previous 10 years and had not received any antibiotic therapy during the previous year. At admission, she was screened for VRE colonization with a positive result; during a retrospective analysis of the antibiotic susceptibility of all VRE strains isolated from infected and/or colonized patients admitted to the ICU, this patient’s strain was found to be LRVRE. She had no signs of VRE infection at presentation and did not develop VRE infection during hospitalization. The General Hospital of Verona is a 1,200-bed tertiarycare hospital. The hospital-wide ICU has 18 beds and in 2004 it registered a total of 766 admissions. In the same year, 127 patients of this ICU were enrolled in a survey of VRE colonization. Rectal swab or fecal samples were inoculated into supplemented Columbia colistin-nalidixic acid broth (bioMérieux, Rome, Italy). After 24 h of incubation at 35°C, a subculture was performed on bileesculin azide solid medium (bioMérieux) supplemented with vancomycin (6 μg/ml); plates were incubated at 35°C and examined at 24 and 48 h. On the basis of the growth observed, isolates demonstrating colony morphology consistent with that of enterococci were identified using API Strep ID 32 identification strips (bioMeriéux). Antibiotic susceptibility testing was performed using the standardized disk diffusion method [2] or the E-test (AB Biodisk, Milan, Italy). MIC interpretative standards of the Clinical and Laboratory Standards Institute (formerly NCCLS) for Enterococcus spp. tested against linezolid were as follows: <2 μg/ml for the susceptible category, 4 μg/ml for the intermediate category and >8 μg/ml for the resistant category [2]. E. faecalis ATCC 29212 was used for quality control. In the patient group enrolled in the survey, a prevalence of 24% was found for patients colonized and/or infected by VRE, and a retrospective analysis of linezolid susceptibility carried out on these strains showed that 11.5% of patients were colonized and/or infected by VRE strains that M. G. Bonora . M. Ligozzi Dipartimento di Patologia, Sezione di Microbiologia, Università di Verona, Strada le Grazie 8, 37134 Verona, Italy

Resistance of Enterococci to Ampicillin and Glycopeptide Antibiotics in Italy

We report the results of the monitoring of resistance to ampicillin, vancomycin, and teicoplanin in enterococci by an Italian network of clinical microbiology laboratories. A total of 16,226 strains were analyzed; 9,169 of these strains were Enterococcus faecalis, 913 were Enterococcus faecium, and 6,144 were Enterococcus species. The average rate of resistance to ampicillin was 1.9% (range, 0-6.5%) for E. faecalis and 70% (range, 33.3%-98.7%) for E. faecium; the average rate of resistance to vancomycin was 1.1% (range, 0.1%-2%) for E. faecalis and 8.5% (range, 0-36.1%) for E. faecium; and the average rate of resistance to teicoplanin was 0.8% (range, 0-2.4%) for E. faecalis and 8.7% (range, 0-36.1%) for E. faecium.