G. Ravizzola

P. aeruginosa bloodstream infections among hematological patients: an old or new question?

Pseudomonas aeruginosa is a well-known cause of severe and potentially life-threatening infections among hematological patients. A prospective epidemiological surveillance program ongoing at our Hematology Unit revealed an increase over time of P. aeruginosa bloodstream infections (BSI). Their impact on outcome and antibiotic susceptibility was analyzed. BSI which consecutively occurred at our institution during a 70-month period were evaluated and correlated with type of pathogen, status of underlying disease, neutropenia, previous antibiotic therapy, resistance to antibiotics, and outcome. During the observation period, 441 BSI were recorded. Frequency of Gram-negative BSI was higher than that of other pathogens (57.3%). Overall, 66 P. aeruginosa BSI were recorded; 22 out of 66 were multiresistant (MR P. aeruginosa). Thirty-day mortality for all BSI was 11.3%; it was 27.3% for P. aeruginosa BSI and 36.4% for MR P. aeruginosa. At multivariate analysis, only active hematological disease and P. aeruginosa BSI were associated to an increased risk of death. For MR P. aeruginosa, BSI mortality was 83.3% vs. 18.8% when empiric therapy included or not an antibiotic with in vitro activity against P. aeruginosa (p = 0.011). Together with active disease, the emergence of P. aeruginosa BSI, particularly if multiresistant, was responsible for an increased risk of death among hematological patients at our institution. In this scenario, reconsidering the type of combination antibiotic therapy to be used as empiric treatment of neutropenic fever was worthwhile.

Recent increase in enterococci, viridans streptococci, Pseudomonas spp. and multiresistant strains among haematological patients, with a negative impact on outcome. Results of a 3-year surveillance study at a single institution

Abstract We prospectively analysed the microbiological isolates of all febrile/infectious episodes occurring at our haematology unit during 2 consecutive 18-month periods. Microbiologically documented infections (MDI) and antibiotic resistance were correlated with type and status of haematological disease, neutropenia, levofloxacin prophylaxis, central venous catheter and clinical outcome. Three hundred and ten MDI were observed and 369 pathogens were isolated. Gram-negative bacteria represented 49.3% and Gram-positive bacteria 40.9% of all pathogens. Fungal infections represented only 8.9% of MDI. A significant decrease in Staphylococcus aureus (p < 0.001) and an increase in enterococci, viridans streptococci and Pseudomonas spp. (p = 0.004) were observed during the second period. Four multiresistant (Multi-R) Pseudomonas were isolated, all during the last 12 months. The death rate in MDI was 8.7%, bacteria accounting for 70.4% of them. Enterococci, streptococci and Pseudomonas spp. infections were involved in 44.4% of MDI with an unfavourable outcome. Multi-R pathogens were involved in 4 cases (3 vancomycin-resistant enterococci and 1 Multi-R Pseudomonas), their death rate being 25%. Multivariate analysis showed that an infection due to a mycotic or a Multi-R pathogen was associated with an unfavourable outcome. The recent emergence of enterococci, viridans streptococci and Pseudomonas spp., particularly if Multi-R, is a major concern in haematological patients.

Bacterial species present in the lower male genital tract: A five-year retrospective study

Objectives To identify bacterial species present in the lower genital tract of males and to investigate the relationship with semen quality. Methods The microscopic analyses and cultures of 696 semen specimens, collected over five years from males investigated for subfertility, were retrospectively assessed. Results Semen cultures were sterile in 48%; they showed a polymicrobial flora (more than two bacterial species) in 30%, and were positive (>1 × 103 colony forming units/ml) in 22% of the cases. Gardnerella vaginalis was the most frequently isolated bacterium, followed by Escherichia coli and Enterococcus sp. Ureaplasma urealyticum was recovered from 13 of 147 samples (9%). Of patients with bacteriospermia 42% had leukospermia (>106 leukocytes/ml of semen). Bacteriospermia and leukospermia did not correlate with each other although a positive correlation was found between the presence of leukocytes and G. vaginalis isolation. Semen parameters were correlated with the bacterial species isolated most frequently. In comparison with controls, sperm concentration, motility and morphology were mostly deteriorated in the presence of G. vaginalis and U. urealyticum. Conclusions Positive seminal fluid cultures must be interpreted with caution, taking into account both raised colony counts of single isolates and leukocyte concentration in the semen. Thus the common misdiagnosis of genital tract infection, based on the presence of seminal bacteria, and unnecessary treatment with antibiotics may be avoided.

Antibiotic resistances and plasmids in Staphylococcus aureus from Italian hospitals

A total of 473 Staphylococcus aureus isolates from six Italian hospitals was examined for susceptibility to several antimicrobial agents and for plasmid content. Methicillin-resistant S. aureus (MRSA) were characterised by a plasmid of mol. wt (10(6)) 18-22 or 25 that carried the determinants for penicillinase production, resistance to cadmium ions and resistance to tetracycline. MRSA isolates usually harboured other smaller plasmids of mol. wt (10(6)) 2.8, 2.6 and 1.65 that encoded resistance to tetracycline, chloramphenicol and erythromycin, respectively, and cryptic plasmids of mol. wt (10(6)) c. 2 and 1 were found frequently. Methicillin-sensitive S. aureus (MSSA) that produced penicillinase often carried plasmids of mol. wt (10(6)) 11 or 13. No particular difference was found in plasmid patterns of strains from the various sources. Analysis of plasmids by EcoRI digestion showed that plasmids of similar mol. wt and phenotypic characteristics may have different restriction patterns, but often share one or more fragments in common.

Prevalence of multidrug-resistant Acinetobacter baumannii and Pseudomonas aeruginosa in an Italian hospital

The severity and extent of disease caused by multidrug-resistant organisms (MDROs) varies by the population(s) affected and the institution(s) at which these organisms are found; therefore, preventing and controlling MDROs are extremely important. A retrospective study of patients who were infected with Acinetobacter baumannii or Pseudomonas aeruginosa was performed at the Spedali Civili Hospital in Brescia, Italy, from 2007 to 2010. A total of 167 (0.52%) A. baumannii isolates and 2797 P. aeruginosa (8.7%) isolates were identified among 31,850 isolates. Amikacin and colistin were the most active agents against A. baumannii strains. Multidrug resistance (MDR) was observed in 57 isolates (54%). Most MDR isolates (42 out of 57, 73%) were resistant to four classes of antibiotics. P. aeruginosa was recovered more frequently from the respiratory tract, followed by the skin/soft tissue, urine and blood. Colistin, amikacin and piperacillin/tazobactam were active against 100%, 86% and 75% of P. aeruginosa isolates, respectively. A total of 20% (n=316) of P. aeruginosa isolates were MDR. In summary, A. baumannii was more rare than P. aeruginosa but was more commonly MDR. Epidemiological data will help to implement better infection control strategies, and developing a local antibiogram database will improve the knowledge of antimicrobial resistance patterns in our region.

Diagnosis of bronchopulmonary infections by quantification of microflora

The quantification of bacteria and fungi in sputum or bronchoaspirate is of clinical value for the diagnosis of respiratory tract infections. We have developed an easy method to count the micro-organisms in patients with respiratory tract infections. This consists of the quantification of micro-organisms by subsequent streakings of a calibrated loop on agar. The correlation between microbiological quantitative data and the clinical status of patients with lower respiratory tract infections is discussed. The data seem to indicate that certain bacteria present in sputum or bronchoaspirate above a certain concentration may be responsible for lower respiratory tract infections. In patients with immunological disorders or chronic pathologies even lower concentrations of micro-organisms in bronchial secretions probably are enough to cause infections. The advantage of this counting method of the microbic species from the respiratory tract consists of their quantification: thus we can attribute an etiological role to a high concentration of the germs, while micro-organisms at low concentrations are probably contaminants. By this method isolated colonies are obtained after 12–18 hours. The bacterial quantification, by respiratory samples examination of the same patient in the following days, allows us to evaluate the efficacy of antibacterial therapy, producing a reduction of bacterial concentration.

Evaluation of the expression of IFN-γ in lymphocytes using a monoclonal antibody and flow cytometry

A stable hybridoma cell line secreting specific antibodies against human interferon-gamma (IFN-gamma) and designated IGMB-14 has been established. It belongs to the IgG1, kappa subclass and it reacts in Western blot with the 28 kDa and 56 kDa polypeptides present in two different affinity purified IFN-gamma preparations. Peripheral blood mononuclear cells (PBMC) from a healthy individual, stimulated in vitro by PHA, were analysed for IFN-gamma production both when viable and following fixation. The presence of cytoplasmic or surface IFN-gamma was visualized by an indirect immunofluorescence assay using monoclonal antibody (MAb) IGMB-14 and a single laser FACS-III fluorescence-activated cell sorter. The staining permitted the detection of newly synthesized cytoplasmic IFN-gamma molecules in lymphocytes at day 1 after PHA stimulation and surface IFN-gamma at day 2. IFN-gamma was expressed on almost all the CD4+ lymphocytes as shown by a double staining technique. The specificity of the reaction was confirmed by Western blots and abolishing IFN-gamma staining by pretreatment of MAb IGMB-14 with IFN-gamma. The presence of surface IFN-gamma was also visualized on freshly isolated PBMC from two patients suffering from measles and AIDS but not on PBMC from a healthy individual. The experiments showed that this immunofluorescent method is useful for the detection, enumeration, and phenotypic characterization of IFN-gamma-producing cells in vitro and, in addition, for evaluating the presence of PBMC expressing IFN-gamma on their surface during a viral disease.

Emergence of the first levofloxacin-resistant strains of Streptococcus agalactiae isolated in Italy

ABSTRACT Of 901 group B streptococcus strains analyzed, 13 (1.4%) were resistant to levofloxacin (MICs of >32 μg/ml for seven isolates, 2 μg/ml for four isolates, and 1.5 μg/ml for four isolates). Mutations in the quinolone resistance-determining regions (QRDRs) of gyrase and topoisomerase IV were identified. A double mutation involving the Ser-81 change to Leu for gyrA and the Ser-79 change to Phe or to Tyr for parC was linked to a high level of fluoroquinolone resistance. In addition, two other mutational positions in parC were observed, resulting in an Asp-83-to-Tyr substitution and an Asp-83-to-Asn substitution. Different mutations were also observed in gyrB, with unknown significance. Most levofloxacin-resistant GBS strains were of serotype Ib and belonged to sequence type 19 (ST19) and clonal complex 19 (CC-19). Most of them exhibited the epsilon gene.

Invasive Candidiasis in Brescia, Italy: Analysis of Species Distribution and Antifungal Susceptibilities During Seven Years

The aims of this study were to evaluate the epidemiology of nosocomial candidemia in a large teaching hospital in Brescia, Italy, and the in vitro antifungal susceptibility of isolates. We analyzed 196 isolates causing fungemia in patients admitted in our hospital, between January 2009 and December 2015. Strains were identified by VITEK 2 and MALDI-TOF MS. MICs were determined by Sensititre Yeast OneTM. The resistance was defined by using the revised CLSI breakpoints/epidemiological cutoff values to assign susceptibility or wild type to systemic antifungal agents. Most infections were caused by Candida albicans (60%), Candida parapsilosis (15%), Candida glabrata (12%) and Candida tropicalis (6%). The susceptibility rate for fluconazole was 96.5%. Non-Candida species isolates exhibited full susceptibilities to echinocandins according to CLSI breakpoints. Amphotericin B demonstrated excellent activity against all Candida species. Local epidemiological and antifungal susceptibility studies are necessary in order to improve empirical treatment guidelines.

A cluster of invasive listeriosis in Brescia, Italy.

Clinical forms were indicated as bacteraemia, miscarriage and peritonitis. Listeriosis was considered hospital-acquired when signs of infection occurred ≥72 h after admission. For each patient with listeriosis, different data were analyzed retrospectively using a medical chart review: demographic information, underlying diseases preceding the onset of listeriosis; days from admission to onset of listeriosis, type of infection and outcome, antibiotic used for therapy. Data on the type of food consumed could not be collected because of insufficient information in medical charts for both the period before admission and during the hospital stay. Before the organism was isolated from microbiological specimens, Listeria had not been considered as being the causative organism in any of these cases. Gram-positive, nonspore forming rods, facultative anaerobes, which produced catalase and a narrow hemolysis, were presumptively identified as Listeria. Further identification was attained using VITEK (bioMérieux, St. Louis, MO, USA) and real-time PCR (Eusepscreen, Eurospital SpA, Trieste, Italy). Genotyping was performed by a multiplex PCR that separates the four major serovars (1/2a, 1/2b, 1/2c, and 4b) of L. monocytogenes strains into four distinct groups as previously described [7]. DNA was extracted from each isolate by the NucliSENS easyMAG (Biomèrieux, Florence, Italy), according to the manufacturer’s instructions. Approximately 1 ng of DNA was used in the PCRs with primers and conditions as described elsewhere [7]. The PCR products were identified by analyzing the unique banding pattern following agarose gel 2 % (wt/vol) electrophoresis. An automated DiversiLab system (BioMérieux), which uses repetitive extragenic palindromic sequencebased PCR (rep-PCR), was performed to further characterize the Listeria isolates. Listeria monocytogenes is a gram-positive facultative intracellular organism that frequently contaminates foodprocessing environments. Increasing incidence rates of invasive listeriosis have been reported over the last few decades by several European countries, with an incidence of listeriosis ranging from 0.3 to 0.8/100,000 people/year in France and Italy, respectively [1]. L. monocytogenes causes an invasive disease with the involvement of central nervous system and bactaeremia with a high case fatality (20– 30 %). Various risk factors have been related to invasive listeriosis. Older adults, where there is an immunosenescence of T-cell-mediated immunity, patients receiving immunosuppressive therapies and people with impaired cell-mediated immunity or autoimmune diseases are at higher risk of invasive listeriosis. Maternal infections are often asymptomatic or mild, but pregnant women can transmit the infection to the fetus, for whom the infection can be fatal [2]. Listeriosis is mainly transmitted through the consumption of contaminated food in the community, and vertical transmission from mother to child. Hospital-associated infections usually occur as clustered outbreaks from environmental contact [3, 4] or contaminated food [5, 6]. Here, we describe a cluster of six cases of invasive listeriosis identified at the Spedali Civili Hospital in Brescia, Italy, between July 2013 and February 2014. A listeriosis case was defined as isolation of L. monocytogenes from a normally sterile site such as blood or cerebrospinal fluid (CSF) or from placental or fetal tissue.