F. Donnarumma

Molecular epidemiological investigation of an outbreak of Pseudomonas aeruginosa infection in an SCT unit

From May to October 2006, six severe Pseudomonas aeruginosa infections were diagnosed in patients undergoing SCT in the SCT unit of the Careggi hospital (Florence, Italy). Four of the infected patients were treated consecutively in the same room (room N). On the hypothesis of a possible environmental source of infection, samples were collected from different sites that had potential for cross-contamination throughout the SCT unit, including the electrolytic chloroxidant disinfectant used for hand washing (Irgasan) and the disinfectant used for facilities cleaning. Four of the environmental samples were positive for P. aeruginosa: three Irgansan soap samples and a tap swab sample from the staff cleaning and dressing room. The AFLP (amplified fragment length polymorphism) typing method employed to evaluate strain clonality showed that the isolates from the patients who had shared the same room and an isolate from Irgasan soap had a significant molecular similarity (dice index higher than 0.93). After adequate control measures, no subsequent environmental sample proved positive for P. aeruginosa. These data strongly support the hypothesis of the clonal origin of the infective strains and suggest an environmental source of infection. The AFLP method was fast enough to allow a ‘real-time’ monitoring of the outbreak, permitting additional preventive measures.

Molecular Characterization of Acinetobacter Isolates Collected in Intensive Care Units of Six Hospitals in Florence, Italy, during a 3-Year Surveillance Program: a Population Structure Analysis

ABSTRACT The strain diversity and the population structure of nosocomial Acinetobacter isolated from patients admitted to different hospitals in Florence, Italy, during a 3-year surveillance program, were investigated by amplified fragment length polymorphism (AFLP). The majority of isolates (84.5%) were identified as A. baumannii, confirming this species as the most common hospital Acinetobacter. Three very distinct A. baumannii clonal groups (A1, A2, and A3) were defined. The A1 isolates appeared to be genetically related to the well-characterized European EU II clone. A2 was responsible for three outbreaks which occurred in two intensive care units. Space/time population dynamic analysis showed that A1 and A2 were successful nosocomial clones. Most of the A. baumannnii isolates were imipenem resistant. The genetic determinants of carbapenem resistance were investigated by multiplex PCR, showing that resistance, independently of hospital origin, period of isolation, or clonal group, was associated with the presence of a bla OXA-58-like gene and with ISAba2 and ISAba3 elements flanking this gene. bla OXA-58 appeared to be horizontally transferred. This study showed that the high discriminatory power of AFLP is useful for identification and typing of nosocomial Acinetobacter isolates. Moreover the use of AFLP in a real-time surveillance program allowed us the recognition of clinically relevant and widespread clones and their monitoring in hospital settings. The correlation between clone diffusion, imipenem resistance, and the presence of the bla OXA-58-like gene is discussed.

Isolation and characterisation of bacterial colonies from seeds and in vitro cultures of Fraxinus spp. from Italian sites

Culturable bacteria were isolated from seeds, embryos and contaminated in vitro cultures of ash (Fraxinus excelsior L., F. ornus L. and F. angustifolia L.) and were identified using morphological and molecular analyses. Fourteen morphologically distinct isolates were recovered from seeds of Fraxinus spp. 16S rDNA sequencing categorised these isolates into ten separate genera. Three strains isolated from contaminated in vitro cultures, Pantoea agglomerans, Staphylococcus succinus and Aerococcus viridans, were used for comparative analysis with isolates from seeds. Antibiotic sensitivity testing of the isolated contaminants, including phytotoxicity of antibiotics on in vitro cultures of ash, was also investigated. Phytotoxic effects on explants immersed in ampicillin or cultured on medium containing ampicillin were negligible, however tetracycline, either alone or in combination with other antibiotics, had phytotoxic effects. We conclude that ampicillin is a suitable antibiotic to limit the growth of contaminating bacteria during the in vitro culture of ash.

Molecular analysis of population structure and antibiotic resistance of Klebsiella isolates from a three-year surveillance program in Florence hospitals, Italy

We report the results of a three-year surveillance program of Klebsiella spp. in six hospitals in Florence (Italy). A total of 172 Klebsiella isolates were identified and typed by AFLP: 122 were K. pneumoniae and 50 were K. oxytoca. Most K. pneumoniae (80%) and K. oxytoca (93%) showed unrelated AFLP profiles. Beside this heterogeneous population structure, we found five small epidemic clonal groups of K. pneumoniae. Four of these groups were involved in outbreak events, three of which occurred in neonatal ICUs. The fifth clonal group spread in three different wards of two hospitals. Only one non-epidemic clonal group of K. oxytoca was detected. The frequencies of isolates with multiple antibiotic resistances increased with time; at the end of the study period, most K. pneumoniae were resistant to all the antibiotics tested. A PCR analysis of seven ertapenem resistant isolates was unable to detect any of the major genes known to underlie carbapenem resistance in K. pneumoniae.

Molecular Surveillance and Population Structure Analysis of Methicillin-Susceptible and Methicillin-Resistant Staphylococcus aureus in High-Risk Wards

ABSTRACT In this study we report the results of analysis of 253 isolates of Staphylococcus aureus (132 methicillin [meticillin]-resistant S. aureus [MRSA] isolates and 121 methicillin-susceptible S. aureus [MSSA] isolates) from 209 patients admitted to 18 high-risk wards of six hospitals located in Florence, Italy, over an 8-month period during which a program of epidemiological surveillance of hospital-acquired infections was conducted. The majority (69%) of the 87 reported S. aureus infections were caused by MRSA. No outbreak events have been reported. All the isolates were typed by amplified fragment length polymorphism (AFLP), and AFLP profiles were analyzed in order to define similarity groups. The discriminatory power of AFLP is very high with MSSA (Simpson index of diversity [D], 95.9%), whereas its resolution capability with MRSA (D, 44.7%) is hampered by the well-known high clonality of these populations (the main MRSA group accounted for 74% of the MRSA isolates). Combining AFLP, improved by visual inspection of polymorphisms, with multiplex PCR greatly increases MRSA resolution (D, 85.5%), resolving the MRSA population to a level that is one of the highest reported in the literature. Widespread and sporadic clones of MSSA and MRSA were identified, and their diffusion in the different hospitals and wards over the surveillance period was studied. The understanding of MSSA and MRSA population structures should be the starting point for the design of a more rational surveillance program for S. aureus species, maximizing benefits and reducing the cost of infection control strategies.