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.

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

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.

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.

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.

Hyper-reactive malarial splenomegaly in a patient with human immunodeficiency virus.

Both hyperreactive malarial splenomegaly (HMS) and HIV infection are highly prevalent in sub-Saharan Africa, but the inter-relationships between the two conditions are not clearly defined. Diagnosis of HMS is particularly difficult in HIV-infected patients, and detection of circulating malaria parasites by polymerase chain reaction (PCR) may represent a useful diagnostic tool.

Carbapenems in the treatment of severe community-acquired pneumonia in hospitalized elderly patients: a comparative study against standard therapy.

In this open, prospective, study were enrolled 204 hospitalized elderly patients with severe (88 males, 116 females, age range 70-94). Patients were randomized to receive one of the following antibiotic treatment regimens: meropenem 500 mg i.v. t.i.d. (52); imipenem/cilastatin 500 mg i.v. t.i.d. (51), clarithromycin 500 mg + ceftriaxone 1 g i.v. b.i.d. (52), clarithromycin 500 mg + amikacin 250 mg i.v. b.i.d. (49). In 99 cases causative germs were isolated (24 meropenem, 26 imipenem, 23 clarithromycin + ceftriaxone, 26 ceftriaxone + amikacin). A satisfactory clinical, bacteriological response was achieved respectively in 86.5% 77% in meropenem; 86.3% 71% in imipenem/cilastatin; 69% 61% in ceftriaxone + clarithromycin and in 85.7% 77% in clarithromycin + amikacin. The mean total cost for each patient was $1,560; $1,620; $1,760 and $1,792 in meropenem, imipenem/cilastatin, clarithromycin + ceftriaxone and clarithromycin + amikacin respectively. This study shows that treatment with either meropenem or imipenem is as efficacious as conventional therapy in the treatment of community acquired pneumonia (CAP), and that meropenem is the most cost-effective.

In-vitro study of the synergy between beta-lactam antibiotics and glycopeptides against enterococci.

The synergy between glycopeptides and beta-lactams was studied using different techniques such as broth macrodilution, killing curves and agar dilution combined with agar diffusion. Two glycopeptide-resistant enterococci isolated from different clinical samples were used. Results showed different effects with significant changes in MICs. Antibacterial activity was related to the concentration of glycopeptide and beta-lactam for Enterococcus faecalis 8253, while for Enterococcus faecium 8072 a paradoxical effect was observed. With this strain, the best synergic effect was detected at teicoplanin concentrations of 1-4 mg/L, but antibacterial activity was reduced at concentrations of 8, 16 and 32 mg/L. No synergic effect was observed with vancomycin. The combination of agar dilution with agar diffusion techniques may constitute a simple method for routine detection of synergic effects between glycopeptides and beta-lactams.

Monoclonal antibodies against antigens exposed on the surface of vegetative forms and spores of Myxococcus virescens.

Twelve monoclonal antibodies (mAbs) directed against cell-surface antigens of Myxococcus virescens cells were developed and partially characterized. All of them recognized multiple, diffuse proteic bands in Western blot and four were also reactive to living bacteria, as assessed by flow cytometry. The four latter mAbs recognized antigens common to a number of vegetative forms and spores. The selective expression of proteins recognized by mAbs on the microorganisms and the possible applications of mAbs to the study of myxobacterial cell interaction are discussed.

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.

In vitro antibacterial activity of the new quinolone Bay y3118 against clinical isolates.

The in vitro antibacterial activity of the new fluoroquinolone Bay y3118 against 609 clinical isolates was evaluated. Bay y3118 exhibited activity against a broad spectrum of organisms, including Gram-negative bacilli, Gram-positive cocci, mycobacteria. The activity of Bay y3118 was often superior to that of other quinolones. Against Gram-negative bacilli its activity was similar to that of ceftriaxone, cefotaxime, ceftazidime and imipenem except for Serratia marcescens, Klebsiella pneumoniae, Enterobacter spp. and Xanthomonas maltophilia, where its activity was superior. Gentamicin and piperacillin sometimes were less active. Bay y3118 was active against a large number of Gram-positive cocci. The fluoroquinolones tested were active against all the strains of Mycobacterium tuberculosis, but only Bay y3118 was effective against Mycobacterium avium.

A multicentre study: Staphylococcus and Enterococcus susceptibility to antibiotics.

A multicentre study to evaluate the susceptibility of Gram-positive cocci isolated from clinical samples, was performed by six centres working in different areas of Italy. We examined 4,544 strains of Staphylococcus aureus, 4,381 strains of coagulase-negative staphylococci and 2,478 strains of enterococci. The following antibiotics were tested: penicillin G, ampicillin, amoxicillin, piperacillin, imipenem, oxacillin, ofloxacin, pefloxacin, ciprofloxacin, gentamicin, tobramycin, amikacin, netilmicin, rifampicin, clindamycin, tetracycline, cotrimoxazole, erythromycin, chloramphenicol, vancomycin and teicoplanin. Oxacillin-susceptible staphylococci confirmed their susceptibility to many other antimicrobial agents while oxacillin-resistant strains confirmed their multiple and frequent resistance to antibiotics. Resistance to oxacillin, cotrimoxazole and chloramphenicol was more frequent in coagulase-negative staphylococci than in Staphylococcus aureus. Aminoglycosides, rifampicin and quinolones were more active against coagulase-negative staphylococci than against Staphylococcus aureus. Enterococci were susceptible to penicillins and imipenem, and moderately susceptible to ciprofloxacin. Susceptibility of 70-79% was observed with high levels of aminoglycosides. Excellent results against staphylococci and enterococci were observed with vancomycin and teicoplanin.

Effect of subinhibitory concentrations of lomefloxacin on bacterial adherence.

We studied the effect of subinhibitory concentrations of lomefloxacin on bacterial adherence, assessing the presence of fimbriae in E. coli and the adherence to uroepithelial cells of Staphylococcus saprophyticus. The degree of inhibition of fimbriae in E. coli in the log-phase is directly proportional to subminimal inhibiting concentrations (sub-MIC). On the contrary, there is no effect when the bacteria are in a stationary phase. In the case of Staphylococcus saprophyticus the adherence to epithelial cells was observed at concentrations of 1/2 and 1/4 the MIC.

Development of a flow cytometric assay for the detection and measurement of neutralizing antibodies against human immunodeficiency virus.

Cells infected with human immunodeficiency virus type 1 (HIV-1) develop viral antigens which can be detected by immunofluorescence. We developed a flow cytometric immunofluorescence assay (FIFA) which provides a quantitative analysis of HIV-1 p24 using a monoclonal antibody (mAb) as a specific reagent. The reduction of HIV p24 antigen expression in viral infected cells was then used to determine HIV neutralizing antibody titers in human sera. Results obtained by FIFA for detecting neutralizing antibodies against HIV-1, when compared with results obtained by indirect immunofluorescence assay (IFA), showed an overall index of agreement of 94.1%. The correlation between the neutralizing antibody titers obtained with each method was found to be highly significant (ro = 0.8; p < 0.01). The simple methodology and the adaptability of this FIFA to various assay conditions make it suitable for routine purposes and for assessing the efficacy of vaccination strategies.

Rapid methods for microbial diagnosis of mycobacterial infections.

The Authors describe their experience in rapid diagnosis of mycobacterial infections using a combination of a radiometric blood culture (Bactec 13 A) and a nucleic acid hybridization system (Gen probe, Accuprobe) to detect and identify Mycobacteria. They found out that a high number of septicaemias in HIV positive patients are due to Mycobacterium avium, while in HIV negative subjects Mycobacterium tuberculosis is the most frequent mycobacterium.

A multicentre study to evaluate the effect of sulbactam/ampicillin combination on anaerobic micro-organisms.

Ampicillin combined with the beta-lactamase inhibitor sulbactam was compared with ampicillin alone, cefoxitin and metronidazole against 569 clinical strains of anaerobic organisms. The strains included 289 species of Bacteroides, 160 strains of Clostridium and 120 strains of various species of Streptococcus/Peptostreptococus, Fusobacterium, Veillonella, Eubacterium, Bifidobacterium, Actinomyces and Propionibacterium. Sulbactam/ampicillin was as effective as cefoxitin and metronidazole against all anaerobic species tested, inhibiting more than 90% of strains at the breakpoints (16, 32 and 32 mg/l, respectively). Sulbactam/ampicillin was also significantly more active than ampicillin against strains of Bacteroides, the minimal inhibitory concentration being at least four-fold lower. In contrast, the activity of the combination did not differ from that of ampicillin alone against Fusobacterium species and Gram-positive rods and cocci.

Antibacterial activity of the new carbapenem meropenem (SM-7338) against clinical isolates.

The in vitro antibacterial activity of the new carbapenem antibiotic meropenem (SM-7338) against 567 clinical isolates was evaluated. SM-7338 exhibited activity against a broad spectrum of organisms, including aerobes and anaerobes, and was superior to the other beta-lactam drugs tested (piperacillin, cefotaxime, ceftazidime, ceftriaxone, cefoxitin). SM-7338 was more active than imipenem, gentamicin and amikacin against Enterobacter cloacae and Pseudomonas aeruginosa. SM-7338 was less potent than imipenem against staphylococci and enterococci, but the activity of the two antibiotics against anaerobes was similar. SM-7338 and imipenem showed a high bactericidal activity at a concentration of 2-4 x MIC.

In-vitro activity of meropenem against clinical isolates in a multicentre study in Italy.

A multicentre in-vitro study was undertaken to evaluate the susceptibility of bacterial pathogens isolated in different Italian hospitals to meropenem. A total of 1399 aerobic and 452 anaerobic strains was analysed. Comparative agents were imipenem, cefotaxime, ceftazidime, ceftriaxone, piperacillin, ciprofloxacin, gentamicin, amikacin, plus vancomycin when appropriate. The MIC ranges (mg/l) of meropenem were: 0.015-2 for Klebsiella spp., Proteus spp., Morganella morganii and Providencia spp.; less than 0.008-1 for Escherichia coli; 0.016-32 for Serratia spp.; 0.03-2 for Enterobacter spp. and Citrobacter spp.; 0.03- greater than 128 for Acinetobacter anitratus; 0.03-32 for Pseudomonas spp.; less than 0.008-0.5 for Haemophilus spp. and Neisseria spp.; 0.015-64 for Staphylococcus spp.; 0.06- greater than 128 for Enterococcus spp.; less than 0.008-0.25 for Streptococcus spp.; 0.016-8 for Fusobacterium spp.; 0.03-8 for Bacteroides spp.; less than 0.06-0.5 for anaerobic Gram-positive cocci; 0.08-2 for Clostridium spp. Meropenem exhibited superior antibacterial activity against the aerobic and anaerobic strains tested when compared to the other beta-lactam drugs. The new carbapenem was as active as ciprofloxacin and more active than imipenem and the aminoglycosides against Enterobacteriaceae and Ps. aeruginosa. It was also more active than ciprofloxacin against most strains of Gram-positive cocci. Meropenem was slightly less potent than imipenem against staphylococci and enterococci, with the exception of oxacillin-susceptible Staph. aureus against which meropenem and imipenem exhibited similar antibacterial activity.