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Background: For years, Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae have represented a serious health problem in hospitals worldwide. Since its approval in 2015, ceftazidime-avibactam (CAZ-AVI) had been successfully used for treating complicated KPC-K. pneumoniae infections, until increasing reports of resistance began to emerge. Methods: Phenotypic tests and molecular analysis were performed in four multidrug-resistant K. pneumoniae isolates, collected from two patients following treatment with CAZ-AVI. Results: In this study, we report two cases of emergence of CAZ-AVI resistance in KPC-3-producing K. pneumoniae isolates, collected from two patients following treatment with CAZ-AVI. Molecular analysis highlighted the D179Y mutation in the bla KPC-3 gene, whose role in the loss of hydrolytic activity (resulting in decreased carpabenem minimum inhibitory concentrations and negative phenotypic tests) of the enzyme has already been shown. Conclusion: Most surveillance schemes aimed at detecting carbapenem-resistant Enterobacteriaceae (CRE) rely on confirmatory phenotypic tests for detecting carbapenemase production. As reports of these treatment-induced, altered CRE phenotypes are increasing, the initial susceptibility testing should be followed by a combination of phenotypic and molecular methods, to make sure that no potential carbapenemase-producing bacteria are missed.

Setting Timely diagnosis of tuberculosis (TB) is essential for effectively controlling and managing the disease. Although international guidelines recommend acid-fast bacilli staining and culture as the 'gold standard', new molecular methods are available to safely and rapidly identify positive samples. Objective To evaluate the performance of the newer and fully automated version of a molecular assay for rRNA amplification (TRCReady® M.TB) on 1028 respiratory samples collected from 378 patients for its possible use as a reliable screening method. Results were evaluated using culture as the reference test. Results Of four diagnostic protocols employed, best results were obtained when TRCReady M.TB was used together with microscopy on the first respiratory sample, followed by microscopy alone on a second one. The sensitivity and specificity were respectively 97% and 100%, with a turnaround time of 24 h. We propose a possible laboratory algorithm for rapid identification of patients with TB. Conclusions TRCReady offers the advantages of full automation and avoidance of cross-contamination. As such, it should be considered as a more economical option for TB screening than other commercial assays that are currently available.

Equine pulmonary aspergillosis is a rare deep mycosis often due to the hematogenous spread of hyphae after gastrointestinal tract disease. We describe herein the main clinic-pathological findings observed in a foal, which spontaneously died after showing diarrhea and respiratory distress. Necropsy and histopathological investigations allowed to diagnose pulmonary aspergillosis, which likely developed after necrotic typhlitis-colitis. Biomolecular studies identified Aspergillus section Fumigati strain as the causative agent. Notably, severe oxalate nephrosis was concurrently observed. Occasionally, oxalate nephropathy can be a sequela of pulmonary aspergillosis in humans. The present case report suggests that the renal precipitation of oxalates can occur also in horses affected by pulmonary aspergillosis and could likely contribute to the fatal outcome of the disease.

Objectives Clostridioides difficile infection (CDI) represents a challenging issue, with an evolving epidemiology. Main objectives of our study were: to assess the frequency of diarrhea of overall etiology, including CDI, as a cause of hospital admission or occurring during hospital stay;- to determine the rate of underdiagnosis of community-acquired (CA-), health care associated (HCA)- and hospital onset (HO-) CDI, and explore factors associated with its clinical suspicion by physicians. Methods A prospective cohort study included all hospitalized patients with diarrhea at two acute-care hospitals. C. difficile (CD) tests were performed on every stool samples, irrespective of the treating physician request. Factors associated with the likelihood of CD test request by physicians were assessed. Results We enrolled 871 (6%) patients with diarrhea. CD test performed on all diarrheic stool samples was positive in 228 cases (26%); 37, 106, 85 cases of CA- (14%), HCA- (42%) and HO- diarrhea (24%), respectively. Treating physicians did not request CD test in 207 (24%) diarrhea cases. The rate of CDI underdiagnosis was 11% (24/228); it was higher in CA-CDI (27%, 10/37). Logistic regression analysis identified age >65 years (RR 1.1; 95 CI 1.06–1.2) and hospitalizations in the previous 3 months (RR 1.2; 95% CI 1.1–1.3) as independent factors associated with the likelihood of requesting the CD test by the physician. These risk factors differed by epidemiological classification of diarrhea and by hospital. Conclusions Our study confirmed the relevance of CDI underdiagnosis and provided new insights in the factors underlying the lack of CDI clinical suspicion.

Objectives To analyse the strains collected during a 1-year survey of ceftazidime-avibactam-resistant KPC-producing Klebsiella pneumoniae, in order to investigate the molecular mechanisms potentially responsible for their resistant phenotype. Methods Clinical KPC-producing K. pneumoniae isolates were collected from 31 patients in six different hospitals in Rome. For eight of the patients, an additional strain grown before the start of treatment was also available, bringing the total of isolates studied to 39. Antimicrobial susceptibility was determined by automated system, broth microdiluition and E-test as appropriate. In silico analysis of acquired resistance genes was achieved by whole-genome sequencing, while multilocus sequence typing and core genome multilocus sequence typing were employed for molecular typing. Mutations associated with ceftazidime-avibactam resistance were identified by Sanger sequencing of the blaKPC gene. Possible mutations in OmpK35 and OmpK36 outer membrane proteins were also investigated. Results Molecular analyses highlighted the circulation of the ST512, 101 and 307 high-risk clones; 26 of the 31 patients carried a mutated KPC variant, five had a wild-type KPC-3. Among the KPC variants detected, 11 were different mutations within the blaKPC-3 gene, four of which were novel mutational changes. Conclusions Different mutations including single amino-acid substitutions, insertions or deletions within the blaKPC gene were found in 26/31 ceftazidime-avibactam-resistant KPC-producing K. pneumoniae strains belonging to high-risk clones circulating in Italy. Of note, in 14/31 cases the isolates displayed resistance to both ceftazidime-avibactam and carbapenems, raising concerns for the possible selection of a multidrug-resistant phenotype.

In an era of increasing drug resistance and limited numbers of antimicrobials in the drug production pipeline, healthcare-associated infections represent a growing public health threat. When therapeutic options are limited, clinicians often resort to using antimicrobial combinations that produce a synergistic effect on the target pathogen. Novel antibiotics are therefore welcome in the daily practice of medicine. For example, ceftaroline is a broad-spectrum cephalosporin active against a variety of bacteria, including methicillin-resistant Staphylococcus aureus, but with limited activity against enterococci, particularly Enterococcus faecium. In this study, we tested the efficacy of ceftaroline against clinical isolates of gram-positive bacteria (S. aureus, Enterococcus faecalis, and E. faecium) by the broth microdilution and E-test assays, and then evaluated the synergistic effect of ceftaroline and ampicillin using the E-test method. The time-kill assay was used to confirm the data on selected strains. This drug combination has been recently shown to be effective against E. faecalis and could offer the advantage of cost-effectiveness (compared to other synergistic associations) as well as good tolerability. The E-test was chosen because of its relative simplicity of use that makes it suitable for routine clinical laboratories as a quick tool to guide clinicians when confronted with difficult-to-treat infections that may require an empirical approach. Our results indicate the presence of a synergistic effect of ceftaroline and ampicillin on most of the strains used, especially E. faecium and E. faecalis. The fact that two of those Enterococcus strains were vancomycin resistant suggests that the possible use of this combination for combating the spread of vancomycin-resistant enterococci should be explored.

On 31 August, a veterinarian and a farmworker were hospitalised for skin lesions. Both had been exposed to a dead cow on 19 August on a farm near Rome, where eight further cattle died of confirmed anthrax later the same month. At admission, the first case showed a black depressed eschar and another smaller lesion on one hand. The second case presented deep infection of the skin, with involvement of both arms. Anthrax diagnosis was confirmed by detection of B. anthracis DNA in eschar fragments from both patients. T-cell specific immunity was studied by flow cytometry and Elispot assay after stimulation with B. anthracis secretome in blood samples collected from Case 1. Immunoglobulin production was detected by complement fixation assay. In Case 1, specific CD4+ T-cell activation was detected, without antibody production. Specific antibodies were detected only in the second patient with severe cutaneous illness. Both patients recovered. The two human anthrax cases were epidemiologically linked, but anthrax was not suspected at admission in either case. The veterinarian had initially unrecognised professional exposure and the exposed farmworker did initially not report exposure to affected animals. A One Health strategy integrating human and animal investigations was essential to confirm the diagnosis.

Background: For years, Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae have represented a serious health problem in hospitals worldwide. Since its approval in 2015, ceftazidime-avibactam (CAZ-AVI) had been successfully used for treating complicated KPC-K. pneumoniae infections, until increasing reports of resistance began to emerge. Methods: Phenotypic tests and molecular analysis were performed in four multidrug-resistant K. pneumoniae isolates, collected from two patients following treatment with CAZ-AVI. Results: In this study, we report two cases of emergence of CAZ-AVI resistance in KPC-3-producing K. pneumoniae isolates, collected from two patients following treatment with CAZ-AVI. Molecular analysis highlighted the D179Y mutation in the blaKPC-3 gene, whose role in the loss of hydrolytic activity (resulting in decreased carpabenem minimum inhibitory concentrations and negative phenotypic tests) of the enzyme has already been shown. Conclusion: Most surveillance schemes aimed at detecting carbapenem-resistant Enterobacteriaceae (CRE) rely on confirmatory phenotypic tests for detecting carbapenemase production. As reports of these treatment-induced, altered CRE phenotypes are increasing, the initial susceptibility testing should be followed by a combination of phenotypic and molecular methods, to make sure that no potential carbapenemase-producing bacteria are missed.

Objectives Carbapenem-resistant Acinetobacter baumannii (CRAB) is a serious nosocomial pathogen that causes a variety of serious, often life-threatening, infections and outbreaks. This study aimed to investigate the molecular epidemiology of clinical CRAB isolates from an outbreak that occurred in the intensive care unit (ICU) of an Italian hospital. Methods From December 2016 to April 2017, 13 CRAB isolates were collected from seven patients treated in the ICU at ‘L. Spallanzani’ Hospital (Rome, Italy). Typing was performed by repetitive extragenic palindromic PCR (rep-PCR) using a DiversiLab® system. Whole-genome sequencing (WGS) data were used for in silico analysis of traditional multilocus sequence typing (MLST) results, to identify resistance genes and for core genome MLST (cgMLST) analysis. Epidemiological data were obtained from hospital records. Results All isolates showed a carbapenem-resistant profile and carried the blaOXA-23 carbapenemase gene. Typing performed by rep-PCR and MLST showed that the isolates clustered into one group, whilst the cgMLST approach, which uses 2390 gene targets to characterise the gene-by-gene allelic profile, highlighted the presence of two cluster types. These results allowed us to identify two patients who were likely to be the source of two separate transmission chains. Conclusion These results show that WGS by cgMLST is a valuable tool, better suited for prompt epidemiological investigations than traditional typing methods because of its higher discriminatory ability in determining clonal relatedness.

Carbapenem-resistant (CR)Acinetobacter baumanniiis a serious nosocomial pathogen able to cause a variety of serious, often life-threatening infections and outbreaks. We aimed to investigate the molecular epidemiology of clinical isolates of CR-A. baumanniifrom an outbreak occurred in the intensive care unit (ICU) of our hospital.From January to April 2017, 13 CR-A. baumanniiisolates were collected at the “L. Spallanzani” hospital, Rome, Italy; typing was performed by repetitive extragenetic palindromic-based (rep)- PCR-based DiversiLab system and WGS data were used for insilicoanalysis of traditional MLST types, for identifying resistance genes and for core genome multi locus sequence typing (cgMLST) analysis. Epidemiological data were obtained from hospital records.Strains were cultured from 7 patients treated in the ICU of our hospital; all isolates showed a multi-drug resistant (MDR) profile, carrying theblaOXA-23carbapenemase. Typing performed by rep-PCR and MLST showed that the isolates clustered into one group while the cgMLST approach, which uses 2690 gene targets to characterize the gene-by-gene allelic profile ofA. baumannii,highlighted the presence of two cluster types. These results allowed us to identify two patients who entered the ICU already colonized by two different strains of CR-A. baumannii; we hypothesize that these two patients could be the source of two separate transmission chains.Our results show that whole-genome-DNA sequencing by cgMLST is a valuable tool, better suited for prompt epidemiological investigations than traditional typing methods because of its higher discriminatory ability in determining clonal relatedness.

Colonizations due to carbapenem-resistant Enterobacteriaceae (CRE) are a source of antimicrobial resistance transmission in health care settings. Eleven Citrobacter freundii strains producing KPC-3 carbapenemase were isolated from rectal swabs during a 3-year surveillance program. bla KPC-3 -carrying plasmids were found to belong to the IncX3 group in 9 of the 11 strains, and complete nucleotide sequences were obtained for 2 of them. Our results highlight the possible role of C. freundii as reservoir of resistance genes.

Colonizations due to carbapenem-resistant Enterobacteriaceae (CRE) are a source of antimicrobial resistance transmission in health care settings. Eleven Citrobacter freundii strains producing KPC-3 carbapenemase were isolated from rectal swabs during a 3-year surveillance program. blaKPC-3-carrying plasmids were found to belong to the IncX3 group in 9 of the 11 strains, and complete nucleotide sequences were obtained for 2 of them. Our results highlight the possible role of C. freundii as reservoir of resistance genes.

We describe the interspecies transmission of the plasmid-mediated blaKPC-3 gene, which confers carbapenem resistance, between clinically relevant gram-negative bacteria in a single patient. A KPC-3 producing Enterobacter aerogenes was isolated from a hospitalized patient previously colonized and then infected by a Klebsiella pneumoniae ST101 carrying the blaKPC-3 gene. The strains showed identical plasmids. Since intense horizontal exchanges among bacteria can occur in the gut, clinicians should be aware that patients colonized by carbapenem-resistant K. pneumoniae could become carriers of other carbapenem-resistant Enterobacteriaceae.

We recently identified a novel family of macrocyclic amidinoureas showing potent antifungal activity against Candida spp. In this study, we demonstrate the fungicidal effect of these compounds as well as their killing activity in a dose-dependent manner. Transcriptional analysis data indicate that our molecules induce a significant change in the transcriptome involving ATP binding cassette (ABC) transporter genes. Notably, experiments against Candida albicans mutants lacking those genes showed resistance to the compound, suggesting the involvement of ABC transporters in the uptake or intracellular accumulation of the molecule. To probe the mode of action, we performed fluorescence microscopy experiments on fungal cells treated with an ad-hoc synthesized fluorescent derivative. Fluorescence microscopy images confirm the ability of the compound to cross the membrane and show a consistent accumulation within the cytoplasm. Finally, we provide data supporting the in vivo efficacy in a systemic infection murine model setup with a drug-resistant strain of C. albicans.

Candida albicans is among the major agents of mucous membrane mycosis in humans and animals, with systemic and deep infections observed in immunocompromised hosts. We describe a case of fatal granulomatous myocarditis caused by C albicans in a 20-day-old canary (Serinus canaria). The etiologic diagnosis was confirmed by identifying characteristic morphologic features of the organism, combined with histochemical staining, and followed by the use of ad hoc biomolecular analysis.

Infections caused by multidrug-resistant (MDR) Acinetobacter baumannii are a challenging problem worldwide. Here, the molecular epidemiology and the genetic basis of antibiotic resistance in 111 MDR A. baumannii strains isolated from June 2005 to March 2009 from infected patients in 10 intensive care units (ICUs) in central Italy were investigated. Epidemiological typing was performed by random amplification of polymorphic DNA, PCR-based sequence grouping and macrorestriction analysis. MICs of antibiotics were determined by the broth microdilution method. Genes for OXA carbapenemases, metallo-beta-lactamases and the CarO porin were searched for by PCR. Molecular genotyping identified one predominant A. baumannii lineage, related to the international clonal lineage II, accounting for 95.6% of isolates. Isolates referable to this lineage were recovered from all ICUs surveyed and were resistant to nearly all classes of antimicrobials, with the exception of tigecycline and colistin. A high percentage (60.5%) of A. baumannii isolates showed elevated resistance to imipenem (MICs >= 128 mg/L), concomitant with resistance to meropenem. Carbapenem resistance was associated with the presence of either bla(OXA-58)-like (22.8%) or bla(OXA-23)-like (71.1%) carbapenemase genes. Molecular typing showed that the epidemic lineage encoding OXA-23 emerged in 2007 and displaced a genetically related clone encoding OXA-58 that had been responsible for previous ICU outbreaks in the same region. Emergence of the OXA-23 epidemic lineage could result from selective advantage conferred by the bla(OXA-23)-like determinant, which provides increased resistance to carbapenems.

Guazatine is a non-systemic contact fungicide, a mixture of reaction products from polyamines, comprising mainly octa-methylenediamine, iminodi(octamethylene)diamine, octamethylenebis(imino-octamethylene) diamine and carbamonitrile. In this work, the analysis of guazatine mixture by LC and LC-MS has been treated for the first time. In the guazatine mixture diamine derivatives account for 40% of the constituents of guazatine, triamines for 46%, tetramines for 11% and other amine derivatives for 3%. The most abundant individual components are the fully guanidated triamine (GGG, 30.6%) and the fully guanidated diamine (GG, 29.5%) followed by the monoguanidated diamine (GN, 9.8%) and a diguanidated triamine (GGN, 8.1%). The identification and separation of main components of commercial guazatine was performed through a new LC-MS method. The separation was performed on an Alltima C(18) column using linear gradient elution (formic acid in water and acetonitrile) with UV-detection at 200 nm and the identification was performed by ESI(+)-mass spectrometry analysis. The main components (GN, GG, GNG, GGN, GGG and GGGG) were then purified and separated from the mixture. Antimycotic activity of guazatine derivatives was determined on different species and strains belonging to genus Candida. The results obtained suggest that GNG and GGGG components can further be developed in new antifungal compounds with high potential for the treatment of Candida infections.

Background: HIV infection is a risk factor for Clostridium difficile infection (CDI) yet the immune deficiency predisposing to CDI is not well understood, despite an increasing incidence of CDI among such individuals. We aimed to estimate the incidence and to evaluate the risk factors of CDI among an HIV cohort in Italy. Methods: We conducted a retrospective case-control (1:2) study. Clinical records of HIV inpatients admitted to the National Institute for Infectious Disease L. Spallanzani, Rome, were reviewed (2002-2013). Cases: HIV inpatients with HO-HCFA CDI, and controls: HIV inpatients without CDI, were matched by gender and age. Logistic regression was used to identify risk factors associated with CDI. Results: We found 79 CDI episodes (5.1 per 1000 HIV hospital admissions, 3.4 per 10000 HIV patient-days). The mean age of cases was 46 years. At univariate analysis factors associated with CDI included: antimycobacterial drug exposure, treatment for Pneumocystis pneumonia, acid suppressant exposure, previous hospitalization, antibiotic exposure, low CD4 cell count, high Charlson score, low creatinine, low albumin and low gammaglobulin level. Using multivariate analysis, lower gammaglobulin level and low serum albumin at admission were independently associated with CDI among HIV-infected patients. Conclusions: Low gammaglobulin and low albumin levels at admission are associated with an increased risk of developing CDI. A deficiency in humoral immunity appears to play a major role in the development of CDI. The potential protective role of albumin warrants further investigation., Funded by Grant RF 2011-02347608, Ministry of Health.

The aim of this study was to evaluate the in vitro activity of doripenem (DOR) alone and in combination with a variety of commonly used anti-Acinetobacter chemotherapeutic agents against 22 primary multidrug-resistant (MDR) Acinetobacter baumannii isolates (including 17 isolates that were resistant to DOR) from Intensive Care Unit patients. Antibiotic interactions were evaluated using the chequerboard method and the time-kill assay.Considering all antimicrobials in combination with DOR, chequerboard analysis showed synergy in 13 A. baumannii strains (54.2%). Seven strains (29.2%) showed ≥2 synergistic interactions. DOR showed synergy in combination with tigecycline (TIG) (eight strains), colistin (COL) (eight strains), amikacin (AMK) (four strains), ampicillin/sulbactam (two strains), and rifampicin (one strain). Remarkably, synergistic effects were detected only in DOR nonsusceptible strains. Time-kill assays confirmed synergy in eight isolates (giving 10 synergistic interactions) for DOR in combination with TIG (n=4), COL (n=5), and AMK (n=1). No antagonistic interactions were observed with both methods.This study demonstrates the in vitro synergistic activity of DOR in combination with TIG, COL, and AMK against DOR-resistant A. baumannii strains, opening the way to in vivo assessment of novel combination therapies for treatment of infections caused by MDR A. baumannii.

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Carbapenem-resistant Acinetobacter baumannii strains belonging to international clonal lineage II (ICL-II) have become predominant in intensive care units (ICUs) throughout Italy. Between 2005 and 2009, the carbapenem-hydrolyzing class D β-lactamase (CHDL) bla OXA-23 gene became more prevalent than bla OXA-58 among epidemic ICL-II strains showing extensive genetic similarity. These findings posed the question of whether CHDL gene replacement occurred in the homogeneous ICL-II population or a new OXA-23 clone(s) emerged and spread in ICUs. In this study, the changes in the ICL-II A. baumannii population and CHDL gene carriage were investigated in 30 genetically related isolates collected during the bla OXA-58 -to- bla OXA-23 transition period. Pulsotyping, randomly amplified polymorphic DNA (RAPD) analysis, and multilocus sequence typing (MLST) results were combined with multilocus variable-number tandem-repeat (VNTR) analysis (MLVA-8), siderotyping, and plasmid profiling to improve genotype-based discrimination between isolates. Pulsotyping, RAPD analysis, and MLST clustered isolates into a single type. MLVA-8 identified 19 types that clustered into three complexes. All OXA-23-producing isolates formed a single complex, while OXA-58 producers were split into two complexes. Southern blot analysis of the physical localization and genetic context of the CHDL genes showed that bla OXA-58 was invariably located on plasmids, while bla OXA-23 was present within Tn 2006 on the chromosome or both the chromosome and plasmids. These data indicate that the apparently homogeneous population of CHDL-producing ICL-II strains was composed of several independent strains and that, between 2005 and 2009, distinct OXA-23 producers displaced the preexisting OXA-58 producers. Thus, MLVA-8 appears to be a suitable tool not only for investigating A. baumannii population structure but also for high-resolution epidemiological typing.

The population structure of the species Legionella pneumophila was investigated by multilocus variable number of tandem repeats (VNTR) analysis (MLVA) and sequencing of three VNTRs (Lpms01, Lpms04 and Lpms13) in selected strains. Of 150 isolates of diverse origins, 136 (86 %) were distributed into eight large MLVA clonal complexes (VACCs) and the rest were either unique or formed small clusters of up to two MLVA genotypes. In spite of the lower degree of genome-wide linkage disequilibrium of the MLVA loci compared with sequence-based typing, the clustering achieved by the two methods was highly congruent. The detailed analysis of VNTR Lpms04 alleles showed a very complex organization, with five different repeat unit lengths and a high level of internal variation. Within each MLVA-defined VACC, Lpms04 was endowed with a common recognizable pattern with some interesting exceptions. Evidence of recombination events was suggested by analysis of internal repeat variations at the two additional VNTR loci, Lpms01 and Lpms13. Sequence analysis of L. pneumophila VNTR locus Lpms04 alone provides a first-line assay for allocation of a new isolate within the L. pneumophila population structure and for epidemiological studies.

The affordability of modern molecular biology tools and the availability of whole genome sequences have brought substantial improvement in research on pathogenic fungi and diagnosis of fungal infection. Molecular methods have resolved many critical aspects of mycological diagnosis by (1) providing species-level identification of fungi through sequencing of suitable taxonomic markers; (2) shortening of the time required for microbiological confirmation of life-threatening fungal infections; and (3) tracing the molecular epidemiology of fungal diseases. Nucleic acids-based methods are less subjective than microscopy- or culture-based methods and unaffected by fungal growth conditions, thus capable of discriminating between phenotypically undistinguishable species. This chapter focuses on the contribution of DNA-based techniques to the identification of clinically important fungi such as Aspergillus, Blastomyces, Candida, Coccidioides, Cryptococcus, Dematiaceous fungi, Fusarium, Histoplasma, Trichosporon, Zygomycetes, and Dermatophytes. Because of their excellent performances, molecular assays are being increasingly adopted by clinical laboratories to complement conventional methods, providing new diagnostic capabilities.

Two epidemiologically unrelated carbapenem-resistant Acinetobacter baumannii isolates were investigated as representatives of the first Italian isolates producing the OXA-24 carbapenemase. Both isolates were of European clonal lineage II and carried an identical OXA-24-encoding plasmid, named pABVA01. Comparative analysis revealed that in pABVA01, bla OXA-24 was part of a DNA module flanked by conserved inverted repeats homologous to XerC/XerD binding sites, which in other Acinetobacter plasmids flank different DNA modules, suggesting mobilization by a novel site-specific recombination mechanism.

Sir, Acinetobacter baumannii has emerged as a leading nosocomial pathogen, particularly in intensive care units (ICUs), where several outbreaks have been described. Treatment of severe A. baumannii infection represents a difficult clinical challenge due to the propensity of this organism to acquire antimicrobial resistance, resulting in the emergence of multiand pan-resistant clones. There are few therapeutic options to combat multidrug-resistant (MDR) A. baumannii. Tigecycline, a new semi-synthetic tetracycline, has provided hope for the treatment of bacterial infections. Literature data on the in vitro activity of tigecycline against MDR A. baumannii shows variable susceptibility, – 7 likely resulting from different susceptibility testing methods and high clonal correlation among epidemic strains. Indeed, early studies have reported excellent in vitro activity of tigecycline against isolates of the A. baumannii complex (MIC90 2 mg/L). 3 However, A. baumannii isolates showing reduced susceptibility to tigecycline (MIC90 32 mg/L) have recently been identified, posing the risk for selection and spreading of resistance in this notoriously adaptable species. Therefore, we investigated the in vitro activity of tigecycline against a collection of 80 presumed MDR A. baumannii isolates and compared the activity between tigecycline and 17 other antimicrobials, including three tetracyclines. A. baumannii primary isolates were obtained between January 2004 and June 2005 from ICU patients cared for in six general hospitals of the Rome (Italy) urban area. None of the patients had undergone previous treatment with tigecycline. Bacteria were identified by the Vitek II system (bioMerieux, Marcy l’Etoile, France) and Amplified Ribosomal DNA Restriction Analysis. Although all isolates had been provided as MDR by collaborating centres, antimicrobial susceptibility testing was re-assessed using ampicillin/ sulbactam, piperacillin, piperacillin/tazobactam, cefepime, ceftazidime, aztreonam, imipenem, meropenem, amikacin, gentamicin, ciprofloxacin, levofloxacin, trimethoprim/sulfamethoxazole, colistin, tetracycline, doxycycline, minocycline and tigecycline. For all drugs, except colistin, doxycycline, minocycline and tigecycline, susceptibility tests were performed by the Vitek II ID-GNB and AST-GN09 cards. Doxycycline and minocycline susceptibility was determined by the disc diffusion method. Tigecycline and colistin susceptibility was determined by the broth microdilution method, according to the CLSI guidelines. The US FDA breakpoints approved for Enterobacteriaceae were applied to define tigecycline susceptibility ( 2 mg/L susceptibility; 8 mg/L resistance). Colistin susceptibility was interpreted according to Gales et al. The MDR phenotype was defined as diminished susceptibility to more than one of the following five drug classes: antipseudomonal cephalosporins, antipseudomonal carbapenems, b-lactam/b-lactamase inhibitor combinations, antipseudomonal fluoroquinolones and aminoglycosides. The in vitro activity of individual antimicrobial agents against A. baumannii clinical isolates is summarized in Table 1. Seventy-eight isolates were definitively defined as MDR. The most common phenotype ( 90% of the isolates) was resistant to piperacillin, piperacillin/tazobactam, cefepime, ceftazidime, aztreonam, ciprofloxacin and levofloxacin. Individual resistance to imipenem, amikacin, trimethoprim/sulfamethoxazole and tetracycline was observed in 70% to 86.2% of the isolates. Approximately 50% of the A. baumannii isolates were resistant to gentamicin and 22.5% to ampicillin/sulbactam. A high level of carbapenem resistance was observed, with 77.5% and 81.3% of isolates resistant to imipenem and meropenem, respectively. Concerning tigecycline, 27.5% of the isolates were nonsusceptible, including 23.7% intermediate and 3.7% fully resistant isolates. The MIC50 and MIC90 values were 2 and 4 mg/L, respectively, with a unimodal distribution of tigecycline MICs in the 0.125–8 mg/L range. These findings are in the middle-upper range of previously reported tigecycline resistance data.3 – 7 Notably, the only isolate resistant to colistin was susceptible to tigecycline. Recent data on the activity of tetracyclines against A. baumannii are conflicting. In large surveys from the UK, the USA, Germany and Italy, the frequency of A. baumannii isolates resistant to tigecycline, minocycline and doxycycline varied in the ranges 4% to 6%, 0% to 18% and 6% to 44%, respectively, in association with high overall rates of resistance to tetracycline. – 7 Conversely, a study conducted in Israel showed that 66% and 37% of the MDR A. baumannii isolates were resistant to tigecycline and minocycline, respectively. Here, we report that 3 (3.8%), 13 (16.3%) and 69 (86.3%) out of the 80 presumed MDR A. baumannii isolates were resistant to minocycline, doxycycline and tetracycline, respectively. Taking into account non-susceptible isolates (intermediate and fully resistant), the relative activities of tetracyclines were minocycline . tigecycline doxycycline . tetracycline, with an overall decreased susceptibility to tigecycline among A. baumannii isolates resistant to other tetracyclines. In fact, all isolates susceptible to minocycline, doxycycline and tetracycline were highly susceptible to tigecycline (MIC range 0.125–1 mg/L), whereas only 6 of 16 (37.5%) isolates non-susceptible to the three tetracyclines were susceptible to tigecycline (MIC range Journal of Antimicrobial Chemotherapy (2008) 62, 422–425 doi:10.1093/jac/dkn172 Advance Access publication 19 April 2008

The utility of a genotypic typing assay for Legionella pneumophila was investigated. A multiple-locus variable number of tandem repeats (VNTR) analysis (MLVA) scheme using PCR and agarose gel electrophoresis is proposed based on eight minisatellite markers. Panels of well-characterized strains were examined in a multicenter analysis to validate the assay and to compare its performance to that of other genotyping assays. Excellent typeability, reproducibility, stability, and epidemiological concordance were observed. The MLVA type or profile is composed of a string of allele numbers, corresponding to the number of repeats at each VNTR locus, separated by commas, in a predetermined order. A database containing information from 99 L. pneumophila serogroup 1 strains and four strains of other serogroups and their MLVA profiles, which can be queried online, is available from http://bacterial-genotyping.igmors.u-psud.fr/ .

Extranuclear or nongenomic effects of thyroid hormones do not require interaction with the nuclear receptor, but are probably mediated by specific membrane receptors. This review will focus on the extranuclear effects of thyroid hormones on plasma membrane transport systems in non mammalian cells: chick embryo hepatocytes at two different stages of development, 14 and 19 days. At variance with mammals, the chick embryo develops in a closed compartment, beyond the influence of maternal endocrine factors. Thyroid hormones inhibit the Na+/K+-ATPase but stimulate the Na+/H+ exchanger and amino acid transport System A with different dose-responses: a bell-shaped curve in the case of the exchanger and a classic saturation curve in the case of System A. These effects are mimicked by the analog 3,5-diiodothyronine. Signal transduction is mediated by inter-play among kinases, mainly protein kinase C and the MAPK pathway, initially primed by second messengers such as Ca2+, IP3, and DAG as in mammalian cells. Thyroid hormones and 3,5-diiodothyronine stimulate thymidine incorporation and DNA synthesis, associated with the increased levels and activity of cyclins and cyclin-dependent kinases involved in the G1/S transition, and also these effects have their starting point at the plasma membrane. Increasing evidence now demonstrates that thyroid hormones act as growth factors for chick embryo hepatocytes and their extranuclear effects are important for prenatal development and differentiation. (c) 2005 Elsevier Inc.

L-T3 and L-T4 activated the Na+/H + exchanger of L-6 myoblasts, with a fast nongenomic mechanism, both in the steady state and when cells undergo acid loading with ammonium chloride. Monitored with the intracellular pH-sensitive fluorescent probe 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein, activation of the exchanger appeared to be initiated at the plasma membrane, because T 3-agarose reproduced the effect of L-T3, and triiodothyroacetic acid, a hormone analog previously shown to inhibit membrane actions of thyroid hormone, blocked the action of L-T3 on the exchanger. We show here for the first time that transduction of the hormone signal in this nongenomic response requires tyrosine kinase-dependent phospholipase C activation and two different signaling pathways: 1) mobilization of intracellular calcium, assessed by the fluorescent probe fura-2, through activation, of inositol tris-phosphate receptors and without contributions from extracellular calcium or ryanodine receptors; and 2) protein phosphorylation involving protein kinase C and MAPK (ERK1/2), as shown by the use of kinase inhibitors and by immunoblotting for activated kinases. Fil: D'Arezzo, Silvia. Università di Roma; Italia Fil: Incerpi, Sandra. Università di Roma; Italia Fil: Davis, Faith B.. Ordway Research Institute; Estados Unidos Fil: Acconcia, Filippo. Università di Roma; Italia Fil: Marino, Maria. Università di Roma; Italia Fil: Farias, Ricardo Norberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Davis, Paul J.. Ordway Research Institute; Estados Unidos

Low physiological concentrations of 17beta-estradiol increased the intracellular pH of rat aortic smooth muscle cells by a rapid nongenomic mechanism. This effect was due to stimulation of the Na+/H+ exchanger activity, measured using the intracellular pH-sensitive fluorescent probe 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. The 17beta-estradiol gave rise to a bell-shaped dose response, with a maximum at 10-12 m and no significant effect at 10-9 m. The specificity of the effect was verified by the use of the Na+/H+ exchanger inhibitor 5-(ethyl-N-isopropyl)amiloride and the lack of effect of the isomer 17alpha-estradiol. Inhibitors of the nuclear estrogen receptors, tamoxifen and ICI 182,780, completely prevented activation of the exchanger by 17beta-estradiol. The effect of low estrogen concentrations on the intracellular pH was mimicked by both norepinephrine and phenylephrine, suggesting a connection between the increase of intracellular pH and the muscle contraction process. The transduction mechanism for this nongenomic effect of estrogens did not involve modulation of the cAMP content, whereas inositol 1,4,5-trisphosphate, protein kinase C and MAPK pathways appear to play a role, as indicated by both pharmacological approaches and immunoblot experiments on protein kinase C translocation and ERK phosphorylation. These results for the first time provide evidence for a nongenomic effect of low physiological concentrations of 17beta-estradiol on intracellular pH that, together with other factors, may contribute to the development of hypertension and atherosclerosis in men and postmenopausal women and increase the risk of cardiovascular disease. Paradoxically, the lack of stimulation at high physiological estradiol levels could explain the protective effects found in premenopausal women.

Background and methods Pseudomonas aeruginosa is a major infectious threat to immunocompromised patients. We recently reported a fatal epidemic of multidrug-resistant P. aeruginosa in an onchoematology unit, linked to massive contamination of a triclosan-based disinfectant. The aim of this study is to evaluate the antimicrobial activity of triclosan and chlorhexidine digluconate against the epidemic strain of P. aeruginosa, to confirm the hypothesis that the soap dispenser acted as a continuous source of the infection during the outbreak, and to explore the potential role of triclosan in increasing the level of resistance to selected antibiotics. Susceptibility tests and time-kill assays for disinfectans were performed using two commercial formulations containing triclosan and chlorhexidine digluconate, respectively. Antibiotic susceptibility testing was performed by the broth microdilution method. Findings The P. aeruginosa epidemic strain exhibited an extremely high level of triclosan resistance (apparent MIC = 2,125 mg/L), while it was markedly susceptible to chlorhexidine digluconate (apparent MIC = 12.5 mg/L). Upon gradual adaptation to triclosan, the epidemic strain survived for a long period (> 120 h) in the presence of 3,400 mg/L (equivalent to 1.6 × MIC) of triclosan, concomitantly increasing the resistance to six antibiotics that are typical substrates of drug efflux pumps of the resistance nodulation division family. This effect was reversed by efflux pump inhibitors. Conclusions The epidemic P. aeruginosa strain was resistant to triclosan and its previous exposure to triclosan increases antibiotic resistance, likely through active efflux mechanisms. Since P. aeruginosa can become tolerant to elevated triclosan concentrations, the use of triclosan-based disinfectants should be avoided in those healthcare settings hosting patients at high risk for P. aeruginosa infection.