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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.

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.

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.

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.

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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