Elisabetta Mantengoli

Antimicrobial resistance in Europe and its potential impact on empirical therapy

The problem of microbial drug resistance is a major public health concern, due to its global dimension and alarming magnitude, although the epidemiology of resistance can exhibit remarkable geographical variability and rapid temporal evolution. The major resistance issues overall are those related to methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), Enterobacteriaceae producing extended-spectrum beta-lactamases, and multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii. Europe is not free from any of these issues, although their impact may be significantly different in different countries. MRSA rates are high in several European countries, but seem to have levelled off in some settings. Diffusion of VRE is still irregular. The most alarming resistance trends are those observed for Enterobacteriaceae and the Gram-negative non-fermenters, with a generalized increase in rates of resistance to the most important anti-Gram-negative agents (beta-lactams and fluoroquinolones) and the circulation of strains showing multidrug resistance phenotypes.

Antibiotic Usage and Risk of Colonization and Infection with Antibiotic-Resistant Bacteria: a Hospital Population-Based Study

ABSTRACT Accurate assessment of risk factors for nosocomial acquisition of colonization by antibiotic-resistant bacteria (ARB) is often confounded by scarce data on antibiotic use. A 12-month, nested, multicenter cohort study was conducted. Target ARB were methicillin (meticillin)-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and ciprofloxacin-resistant Pseudomonas aeruginosa (CR-PA). Nares and rectal swabs were obtained before and after starting antibiotics. Pulsed-field gel electrophoresis was done to define genetic relatedness of the strains. Primary outcomes were (i) the mean time, in days, for acquisition of target ARB colonization in patients previously not colonized; (ii) the rate of acquisition per 1,000 antibiotic-days according to different classes of antibiotics; (iii) the rate of infection caused by the same bacteria as those previously isolated in screening samples; and (iv) the risk factors for ARB acquisition. In total, 6,245 swabs from 864 inpatients were processed. The rate of acquisition was 3%, 2%, and 1% for MRSA, VRE, and CR-PA, respectively. The rate of acquisition of ARB per 1,000 antibiotic-days was 14 for carbapenems, 9 for glycopeptides, and 6 for broad-spectrum cephalosporins and quinolones. The highest rates of acquisition were observed for carbapenems in dialyzed and diabetic patients. Four risk factors were independently associated with acquisition of target ARB: use of carbapenems, age of >70 years, hospitalization for >16 days, and human immunodeficiency virus infection. During the 30-day follow-up, 4 among 42 patients newly colonized by ARB (9%) suffered from an infection due to the same bacteria as those isolated in a previous screening sample. Colonizing and infecting strains from single patients were genotypically identical. Identifying ARB colonization early during antibiotic therapy could target a high-risk hospitalized population that may benefit from intervention to decrease the risk of subsequent nosocomial infections.

Dynamics of a Nosocomial Outbreak of Multidrug-Resistant Pseudomonas aeruginosa Producing the PER-1 Extended-Spectrum β-Lactamase

ABSTRACT From November 1998 to August 1999, a large outbreak occurred in the general intensive care unit of the Ospedale di Circolo in Varese (Italy), caused by Pseudomonas aeruginosa producing the PER-1 extended-spectrum β-lactamase. A total of 108 clinical isolates of P. aeruginosa resistant to broad-spectrum cephalosporins were recovered from 18 patients. Epidemic isolates were characterized by synergy between clavulanic acid and ceftazidime, cefepime, and aztreonam. Isoelectric focusing of crude bacterial extracts detected two nitrocefin-positive bands with pI values of 8.0 and 5.3. PCR amplification and characterization of the amplicons by restriction analysis and direct sequencing indicated that the epidemic isolates carried a blaPER-1 determinant. The outbreak was of clonal origin as shown by pulsed-field gel electrophoresis analysis. This technique also indicated that the epidemic strain was not related to three other PER-1-positive isolates obtained at the same hospital in 1997. Typing by enterobacterial repetitive intergenic consensus-PCR showed that minor genetic variations occurred during the outbreak. The epidemic strain was characterized by a multiple-drug-resistance phenotype that remained unchanged over the outbreak, including extended-spectrum cephalosporins, monobactams, aminoglycosides, and fluoroquinolones. Isolation of infected patients and appropriate carbapenem therapy were successful in ending the outbreak. Our report indicates that theblaPER-1 resistance determinant may become an emerging therapeutic problem in Europe.

Multifocal Detection of Multidrug-Resistant Pseudomonas aeruginosa Producing the PER-1 Extended-Spectrum β-Lactamase in Northern Italy

ABSTRACT Forty-four nonreplicate clinical isolates of Pseudomonas aeruginosa that were resistant to extended-spectrum cephalosporins (ceftazidime and cefepime) and aztreonam, that putatively produced an acquired extended- spectrum β-lactamase (ESBL), according to the results of a double-disk synergy test, and that had been involved in nosocomial outbreaks were obtained from six different hospitals in northern Italy and screened for the presence of blaPER ESBL determinants. Twenty isolates, associated with nine independent outbreaks that occurred in five hospitals in the Milan area and its surroundings during 1995-2000, were found to carry an acquired blaPER-1 gene. PER-1 producers representative of the nine outbreaks exhibited a multidrug resistance (MDR) phenotype, including resistance to extended-spectrum cephalosporins, aztreonam, meropenem, aminoglycosides, and in most cases, imipenem and ciprofloxacin. An analysis of macrorestriction profiles of their genomic DNAs by pulsed-field gel electrophoresis revealed an overall clonal diversity of the PER-1 producers, although interhospital clonal spread was also observed. The blaPER-1 gene was not transferable and appeared to be chromosomally located. An analysis of the EcoRI and EcoRV restriction fragment length polymorphisms of the blaPER-1 locus revealed identical patterns for all isolates, and the characterization of a 1.9-kb region containing blaPER-1 revealed a conserved structure in representatives of the various clonal lineages. The present findings indicate that MDR P. aeruginosa clones producing the PER-1 ESBL are endemic to this area of northern Italy, where they have been circulating since the mid-1990s and have been associated with several nosocomial outbreaks.

Epidemiology and clinical relevance of microbial resistance determinants versus anti-Gram-positive agents.

Gram-positive pathogens are a major cause of community-acquired and hospital-acquired infections, and exhibit a remarkable ability to develop antibiotic resistance. Methicillin-resistant Staphylococcus aureus (MRSA), glycopeptide-resistant enterococci (GRE) and multidrug-resistant pneumococci are currently the major resistance challenges among Gram-positives, due to their global dissemination and overall clinical impact. The mechanisms of evolution of these resistance phenotypes are based on a diverse array of mutational events and gene transfer phenomena carried out by several types of mobile genetic elements, followed by the dissemination of successful resistant clones. Resistance to glycopeptides in staphylococci remains uncommon, likely due to fitness issues. Resistance to the new anti-Gram-positive agents (linezolid, daptomycin and tigecycline) overall remains very rare. However, a transferable resistance mechanism to linezolid, mediated by ribosomal target modification by the Cfr protein, has recently emerged among S. aureus, being a matter of raising concern. Linezolid resistance among enterococci and coagulase-negative staphylococci is also increasingly reported. Moreover, a role for antibiotic resistance has been advocated in the recent increase of Clostridium difficile infection (CDI) associated with the emergence of hypervirulent strains.

Epidemiology and genetic characteristics of extended-spectrum β-lactamase-producing Gram-negative bacteria causing urinary tract infections in long-term care facilities

OBJECTIVES To assess risk factors for acquiring extended-spectrum β-lactamase-producing Gram-negative bacteria (ESBL+ GN) causing urinary tract infections (UTIs) in long-term care facilities (LTCFs). METHODS A prospective case-case-control study was carried out. In the first study, cases were defined as patients harbouring ESBL+ GN, while, in the second study, cases were defined as patients harbouring ESBL-negative (ESBL-) GN. Controls were selected by simple random sampling from patients without GN infection. ESBL determinants were characterized by hybridization, and confirmed by PCR and sequencing. RESULTS The study involved 297 LTCF patients (99 with ESBL+ GN UTI, 99 with ESBL- GN UTI and 99 without GN infection). ESBL+ GN UTIs were due to Escherichia coli (64%), Proteus mirabilis (25%) and Klebsiella pneumoniae (11%). The CTX-M-type enzymes were the most prevalent (73% of isolates), whereas TEM- and SHV-type ESBLs and AmpC-type enzymes were less prevalent (10%, 2% and 15% of isolates, respectively). Patients with ESBL+ GN UTI were more likely to have a permanent urinary catheter (OR 15, 95% CI 6.9-30.5) and to have received antimicrobial therapy in the previous 30 days (OR 4, 95% CI 1.2-10.9). After adjusting for type, dosage and duration of antibiotic, exposure to ≥7 days of quinolones and third-generation cephalosporins was associated with the highest risk of ESBL+ GN UTI development (OR 7, 95% CI 1.2-40). Independent risk factors for acquiring ESBL- GN UTIs were previous surgical procedures (OR 2, 95% CI 1.1-4) and the presence of a urinary catheter (OR 8, 95% CI 4-16). No specific antibiotics remained a significant risk for ESBL- GN UTI after adjusting for demographic and clinical risk factors. CONCLUSIONS Exposure to ≥7 days of quinolones and third-generation cephalosporins significantly increases the risk of ESBL+ GN UTI. Interventions aimed at improving compliance with antimicrobial stewardship principles should be further developed and implemented in LTCFs.

A novel tetrabranched antimicrobial peptide that neutralizes bacterial lipopolysaccharide and prevents septic shock in vivo

We describe the nonnatural antimicrobial peptide KKIRVRLSA (M33) and its capacity to neutralize LPS‐induced cytokine release, preventing septic shock in animals infected with bacterial species of clinical interest. M33 showed strong resistance to proteolytic degradation when synthesized in tetrabranched form with 4 peptides linked by a lysine core, making it suitable for use in vivo. HPLC and mass spectrometry demonstrated its stability in serum beyond 24 h. M33 was found to be very selective for gram‐negative bacteria. Minimal inhibitory concentration (MIC) ranged from 0.3 to 3 µΜ for multidrug resistant clinical isolates of several pathogenic species, including Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii. M33 neutralized LPS derived from P. aeruginosa and K. pneumoniae, and prevented TNF‐α release from LPS‐activated macrophages, with an EC50 of 3.8e‐8 M and 2.8e‐7 M, respectively, as detected by sandwich ELISA. M33 activity was also tested in sepsis animal models. It averted septic shock symptoms due to Escherichia coli and P. aeruginosa in doses compatible with clinical use (5–25 mg/kg). These properties make tetrabranched M33 peptide a good candidate for the development of a new antibacterial drug.—Pini, A., Falciani, C., Mantengoli, E., Bindi, S., Brunetti, J., Iozzi, S., Rossolini, G. M., Bracci, L. A novel tetrabranched antimicrobial peptide that neutralizes bacterial lipopolysaccharide and prevents septic shock in vivo. FASEB J. 24, 1015–1022 (2010). www.fasebj.org

Tn5393d, a Complex Tn5393 Derivative Carrying the PER-1 Extended-Spectrum β-Lactamase Gene and Other Resistance Determinants

ABSTRACT In Alcaligenes faecalis FL-424/98, a clinical isolate that produces the PER-1 extended-spectrum β-lactamase, the blaPER-1 gene was found to be carried on a 44-kb nonconjugative plasmid, named pFL424, that was transferred to Escherichia coli by electroporation. Investigation of the genetic context of the blaPER-1 gene in pFL424 by means of a combined cloning and PCR mapping approach revealed that the gene is associated with a transposonlike element of the Tn3 family. This 14-kb element is a Tn5393 derivative of original structure, named Tn5393d, which contains the transposition module and the strAB genes typical of other members of the Tn5393 lineage plus additional resistance determinants, including the blaPER-1 gene and a new allelic variant of the aphA6 aminoglycoside phosphotransferase gene, named aphA6b, whose product is active against kanamycin, streptomycin, and amikacin. Tn5393d apparently originated from the consecutive insertion of two composite transposons into a Tn5393 backbone carrying the aphA6b and the blaPER-1 genes, respectively. The putative composite transposon carrying blaPER-1, named Tn4176, is made of two original and nonidentical insertion sequences of the IS4 family, named IS1387a and IS1387b, of which one is interrupted by the insertion of an original insertion sequence of the IS30 family, named IS1066. In pFL424, Tn5393d is inserted into a Tn501-like mercury resistance transposon. Transposition of Tn5393d or modules thereof containing the blaPER-1 gene from pFL424 to small multicopy plasmids or to a bacterial artificial chromosome was not detected in an E. coli host harboring both replicons.

Spread of multidrug-resistant Proteus mirabilis isolates producing an AmpC-type β-lactamase: epidemiology and clinical management

A remarkable increase in Proteus mirabilis strains producing acquired AmpC-type beta-lactamases (CBLs) has been observed at Ospedale di Circolo e Fondazione Macchi (Varese, Italy) over the last few years. The epidemiology and treatment outcome of infections associated with this unprecedented spread are reported. From 2004-2006, 2070 P. mirabilis isolates were investigated. Extended-spectrum beta-lactamases (ESBLs) and CBL resistance determinants were identified by gene amplification and direct sequencing. Clonal relatedness was evaluated by macrorestriction analysis. Overall, 43 CBL-positive isolates were obtained from hospitalised (n=22) and non-hospitalised (n=21) patients (median age 78.8 years). The prevalence of CBL-positive isolates increased from 0.3% in 2004 to 4.6% in 2006, whereas that of ESBL-positive isolates remained constant (ca. 10%). CBL-positive isolates were multidrug-resistant and carried the CMY-16 determinant. All but two isolates were genetically identical or closely related. Retrospective analysis of clinical records revealed that the majority of CMY-16-positive isolates were associated with urinary tract infections. Treatment with amikacin or carbapenems was consistently effective, whereas piperacillin/tazobactam produced a clinical response in seven of nine cases. This is the first report of a rapid spread of CBL-positive P. mirabilis strains endowed with remarkable antimicrobial resistance. Practical methods for CBL detection are needed for the appropriate management of related infections.

Spread in an Italian Hospital of a Clonal Acinetobacter baumannii Strain Producing the TEM-92 Extended-Spectrum β-Lactamase

ABSTRACT Clinical isolates of Acinetobacter baumannii (n = 470) were collected during a 7-year period and investigated for the genetic determinants of resistance to expanded-spectrum β-lactams. Thirty-one isolates produced the TEM-92 extended-spectrum β-lactamase (ESBL) and were clonally related. This is the first report of A. baumannii producing a TEM-type ESBL.