Genomic characterization of Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) strains circulating in three university hospitals in Northern Italy over three years.

OBJECTIVES: Genomic surveillance of Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) is crucial for virulence, drug-resistance monitoring, and outbreak containment. METHODS: Genomic analysis on 87 KPC-Kp strains isolated from 3 Northern Italy hospitals in 2019-2021 was performed by whole genome sequencing (WGS), to characterize resistome, virulome, and mobilome, and to assess potential associations with phenotype resistance and clinical presentation. Maximum Likelihood and Minimum Spanning Trees were used to determine strain correlations and identify potential transmission clusters. RESULTS: Overall, 15 different STs were found; the predominant ones included ST307 (35, 40.2%), ST512/1519 (15, 17.2%), ST20 (12, 13.8%), and ST101 (7, 8.1%). 33 (37.9%) KPC-Kp strains were noticed to be in five transmission clusters (median number of isolates in each cluster: 5 [3-10]), four of them characterized by intra-hospital transmission. All 87 strains harbored Tn4401a transposon, carrying blaKPC-3 (48, 55.2%), blaKPC-2 (38, 43.7%), and in one case (1.2%) blaKPC-33, the latter gene conferred resistance to ceftazidime/avibactam (CZA). Thirty strains (34.5%) harbored porin mutations; of them, 7 (8.1%) carried multiple Tn4401a copies. These strains were characterized by significantly higher CZA minimum inhibitory concentration compared with strains with no porin mutations or single Tn4401a copy, respectively, even if they did not overcome the resistance breakpoint of 8 ug/mL. Median 2 (IQR:1-2) virulence factors per strain were detected. The lowest number was observed in ST20 compared to the other STs (p<0.001). While ST307 was associated with infection events, a trend associated with colonization events could be observed for ST20. CONCLUSIONS: Integration of genomic, resistance score, and clinical data allowed us to define a relative diversification of KPC-Kp in Northern Italy between 2019 and 2021, characterized by few large transmission chains and rare inter-hospital transmission. Our results also provided initial evidence of correlation between KPC-Kp genomic signatures and higher MIC levels to some antimicrobial agents or colonization/infection status, once again underlining WGS's importance in bacterial surveillance.

Microbial composition associated with biliary stents in patients undergoing pancreatic resection for cancer.

Malignant bile duct obstruction is typically treated by biliary stenting, which however increases the risk of bacterial infections. Here, we analyzed the microbial content of the biliary stents from 56 patients finding widespread microbial colonization. Seventeen of 36 prevalent stent species are common oral microbiome members, associate with disease conditions when present in the gut, and include dozens of biofilm- and antimicrobial resistance-related genes. This work provides an overview of the microbial communities populating the stents.

Clinical usefulness of routinely performed drain fluid cultures after pancreaticoduodenectomy. A new ally to predict patients' outcomes?

BACKGROUND: Previous studies showed that bacterial contamination of surgical drains was associated with higher morbidity and mortality following pancreaticoduodenectomy (PD). However, there is still no agreement on the routine use of fluid drainage cultures in the management of patients underwent PD. Therefore, we aimed to clarify the role of surgical drain bacterial contamination in predicting patients' postoperative course. METHOD: Single-centre study including patients underwent PD at Humanitas Research Hospital (2010-2021). Preoperative, intraoperative and postoperative data were collected. Routinely performed fluid drain cultures on postoperative day (POD) 5 were analyzed and compared among patients throughout the cohort. RESULTS: A total of 825 patients were analyzed. Bacterial contamination of surgical drains was observed in 420 (50.9 %) patients and it was found to be associated with a higher rate of B/C grade pancreatic fistula (POPF) (P < 0.001), Clavien-Dindo≥3 (P < 0.001), 30-day mortality (P = 0.011), wound infection (P < 0.001), relaparotomies (P = 0.003) and greater length of hospital stay (LOS) (P < 0.001). Also, E. coli surgical drain contamination was demonstrated to double the risk of B/C grade POPF development (OR = 1.628, 95 % IC = 1.009-2.625, P = 0.046). Finally, preoperative biliary drainage (OR = 2.474, 95 % IC = 1.855-3.298, P < 0.001), age ≥75 years old (OR = 1.492, 95 % IC = 1.077-2.067, P = 0.016) and isolated Roux-en-Y pancreaticojejunostomy (OR = 1.639, 95 % IC = 1.229-2.188, P < 0.001) were identified as risk factors for surgical drains bacterial contamination. CONCLUSION: Bacterial contamination of surgical drains predicts the development of B/C grade POPF and other major complications after PD. Therefore, we suggest the routine use of fluid drain cultures following PD.

Chest CT in patients with a moderate or high pretest probability of COVID-19 and negative swab.

OBJECTIVES: We aimed to assess the diagnostic performance of CT in patients with a negative first RT-PCR testing and to identify typical features of COVID-19 pneumonia that can guide diagnosis in this case. METHODS: Patients suspected of COVID-19 with a negative first RT-PCR testing were retrospectively revalued after undergoing CT. CT was reviewed by two radiologists and classified as suspected COVID-19 pneumonia, non-COVID-19 pneumonia or negative. The performance of both first RT-PCR result and CT was evaluated by using sensitivity (SE), specificity (SP), positive predictive value (PPV), negative predictive value (NPV) and area under the curve (AUC) and by using the second RT-PCR test as the reference standard. CT findings for confirmed COVID-19 positive or negative were compared by using the Pearson chi-squared test (P values < 0.05) RESULTS: Totally, 337 patients suspected of COVID-19 underwent CT and nasopharyngeal swabs in March 2020. Eighty-seven out of 337 patients had a negative first RT-PCR result; of these, 68 repeated RT-PCR testing and were included in the study. The first RT-PCR test showed SE 0, SP = 100%, PPV = NaN, NPV = 70%, AUC = 50%, and CT showed SE = 70% SP = 79%, PPV = 86%, NPV = 76%, AUC = 75%. The most relevant CT variables were ground glass opacity more than 50% and peripheral and/or perihilar distribution. DISCUSSION: Negative RT-PCR test but positive CT features should be highly suggestive of COVID-19 in a cluster or community transmission scenarios, and the second RT-PCR test should be promptly requested to confirm the final diagnosis.

Caspofungin for primary antifungal prophylaxis after T-cell-replete haploidentical stem cell transplantation with post-transplant cyclophosphamide.

OBJECTIVES: T-cell-replete haploidentical stem cell transplantation (Haplo-SCT) with post-transplant cyclophosphamide (PT-Cy) is at high risk of invasive fungal infections (IFI), and anti-mold-active drug is required for primary antifungal prophylaxis (PAP) according to international guidelines. No data are available on the efficacy of caspofungin as PAP in this setting. METHODS: Here, we report our retrospective experience with 103 consecutive patients treated with caspofungin as PAP after Haplo-SCT. Caspofungin was administered only during the pre-engraftment phase. RESULTS: Hundred-day cumulative incidence of proven-probable IFI (PP-IFI) was 8.7% and median day of onset was 19 post-SCT. No patient died of PP-IFI, and overall survival (OS) and non-relapse mortality (NRM) hazard ratio (HR) for patients experiencing IFI were 1.02 (P = 0.9) and 0.7 (P = 0.7), respectively. Three-year overall survival (OS) and 1-year non-relapse mortality (NRM) were 55% and 19%, respectively. By univariate analysis, duration of neutropenic phase and partial remission pre-transplant disease status were associated with increased incidence of IFI, but were not confirmed by multivariate analysis. CONCLUSION: In summary, PAP with caspofungin is an effective strategy for preventing IFI in the context of Haplo-SCT with PT-Cy. Further efforts are required in order to identify more potent strategies able to avoid the occurrence of breakthrough infections.

Efficacy of intraventricular amikacin treatment in pan-resistant Pseudomonas aeruginosa postsurgical meningitis.

BACKGROUND: We describe a case of pan-resistant Pseudomonas aeruginosa postsurgical meningitis associated with the presence of an external ventricular device. We changed therapy twice; finally, by using amikacin and a continuous infusion of cefepime, we obtained clinical improvement. CASE PRESENTATION: A female patient, who underwent surgery for a cavernous angioma, presented with meningitis. Cerebrospinal fluid culture revealed a multidrug-resistant Pseudomonas aeruginosa, initially sensitive only to colistin. We successfully used intrathecal amikacin and intravenous cefepime continuous infusion plus intravenous amikacin after two previous ineffective therapeutic approaches. CONCLUSION: The evaluation of the antibiotic concentration and the bactericidal activity in cerebrospinal fluid may contribute to the choice of the drug in cases of multidrug-resistant meningitis.

A reduced dose of fluconazole as primary antifungal prophylaxis is not associated with increased risk of invasive fungal infections after allogeneic stem cell transplantation from a HLA identical sibling.

BACKGROUND: Invasive fungal infections (IFI) represent a common side effect of allogeneic hematopoietic stem cell transplant (allo-SCT), resulting in increased non relapse mortality (NRM) and reduced overall survival (OS) rates. Seventy-five days of Fluconazole 400 mg/d represents the standard primary antifungal prophylaxis (PAP) after allo-SCT, especially for low-risk transplants. However, the ideal dosage of fluconazole has never been tested. METHODS: Here, we report the experience of our institution on 113 consecutive patients receiving an allo-SCT from a HLA identical sibling between 1999 and 2015, where PAP consisted of fluconazole 100 mg/d only during the pre-engraftment phase. At the time of transplant, all patients were considered at low-risk for mold infection according to ECIL-5 guidelines. RESULTS: Cumulative incidence of possible-probable-proven IFI was 11.7%, while proven-probable (PP-IFI) occurred in 5.5% of patients by day 100 post transplant. Of note, only 1 patient developed invasive Candidiasis due to a non-albicans strain and stool-screening tests were negative for colonization by Candida albicans species. The incidence of 1-year acute and 2-year chronic graft-versus-host-disease (GVHD) was 30% and 45%, respectively. Three-year OS and 1-year NRM were 53% and 11.3%, respectively. CONCLUSION: In summary, fungal prophylaxis with fluconazole 100 mg/d results in very low incidence of PP-IFI, GVHD and NRM in low-risk allo-SCT.

Multicenter prospective study on the prevalence of colistin resistance in Escherichia coli: relevance of mcr-1-positive clinical isolates in Lombardy, Northern Italy.

BACKGROUND: The emergence of the plasmid-mediated colistin resistance mechanism in Escherichia coli has raised concern among public health experts as colistin is a last-line antimicrobial resort. The primary aim of the study was to investigate the prevalence of this resistance trait in E. coli isolates circulating in the Lombardy region, Northern Italy. The presence of mcr-type genes and their genetic relationship were also studied. MATERIALS AND METHODS: A prospective study was performed during a 4-month period (May to August, 2016) in six acute care Hospitals. Consecutive nonduplicate clinical isolates of E. coli from any type of clinical specimen, with the exception of rectal swabs, were included in the study. Isolates that exhibited MIC values for colistin >2 mg/L were further investigated. Bacterial identification was obtained by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Amplification of mcr-type genes (-1 to -5 variants) and microarray analysis were accomplished. Repetitive sequence-based PCR (Rep-PCR) and multilocus sequence typing (MLST) analysis were used for genotyping. RESULTS: Overall, 3,902 consecutive E. coli isolates (2,342 from outpatients, 1,560 from inpatients) were evaluated during the study period. Of them, 18/3,902 (0.5%), collected from 4/6 centers, showed resistance to colistin. These isolates were mostly obtained from urine of both outpatients (n=12) and inpatients (n=6). Colistin MIC values ranged from 4 to 8 mg/L. The mcr-1 gene was detected in 10/18 isolates (7 from outpatients, 3 from inpatients). Rep-PCR and MLST analysis revealed the presence of nine different clusters. Further mcr-type genes were not detected. CONCLUSION: Resistance to colistin in E. coli clinical isolates appears low in our geographic area. With regard to mcr-1-positive isolates, they accounted for approximately 50% of colistin-resistant E. coli isolates, thus representing a relevant resistance mechanism in this context. Although overall limited, the presence of mcr-1 determinant in our region should not be ignored and great concern should be given to the continuous surveillance.

Role of preoperative biliary stents, bile contamination and antibiotic prophylaxis in surgical site infections after pancreaticoduodenectomy.

BACKGROUND: The routine use of preoperative biliary drainage before pancreaticoduodenectomy (PD) remains controversial. This observational retrospective study compared stented and non-stented patients undergoing PD to assess any differences in post-operative morbidity and mortality. METHODS: A total of 180 consecutive patients who underwent PD and had intra-operative bile cultures performed between January 2010 and February 2013 were retrospectively identified. All patients received peri-operative intravenous antibiotic prophylaxis, primarily cefazolin. RESULTS: Overall incidence of post-operative surgical complications was 52.3 %, with no difference between stented and non-stented patients (53.4 % vs. 51.1 %; p = 0.875). However, stented patients had a significantly higher incidence of deep incisional surgical site infections (SSIs) (p = 0.038). In multivariate analysis, biliary stenting was confirmed as a risk factor for deep incisional SSIs (p = 0.044). Significant associations were also observed for cardiac disease (p = 0.010) and BMI ≥25 kg/m(2) (p = 0.045). Enterococcus spp. were the most frequent bacterial isolates in bile (74.5 %) and in drain fluid (69.1 %). In antimicrobial susceptibilty testing, all Enterococci isolates were cefazolin-resistant. CONCLUSION: Given the increased risk of deep incisional SSIs, preoperative biliary stenting in patients underging PD should be used only in selected patients. In stented patients, an antibiotic with anti-enterococcal activity should be chosen for PD prophylaxis.

Multifocal diffusion of a KPC-3 producing ST512 K. pneumoniae clone in Northern Italy.

Sequence Type 258 (ST258) together with its allelic single- and double-locus variants have mostly been associated with the dissemination of KPC-producing Klebsiella pneumoniae in Europe. A total of 56 nonreplicate K. pneumoniae isolates with decreased carbapenem-susceptibility, collected at 7 different hospitals located in Northern Italy were investigated for the occurrence of blaKPC-type genes. PCR and sequencing results highlighted the presence of blaKPC2 or blaKPC-3 determinants in 10/56 and 5/56 cases respectively. Here we describe the intra- and inter-hospital spread in Northern Italy of a K. pneumoniae ST512 clone harboring the blaKPC-3 gene.

Candidemia: species involved, virulence factors and antimycotic susceptibility.

We investigate the characteristics of the Candida species involved in BSI episodes in our Institute, their phospholipase and protease activity and the susceptibility pattern towards the main antifungal agents currently available. From January 2009 to December 2010 we documented a total of 59 episodes of candidemia. The incidence of candidemia was 32% in General Surgery, 22% in the Intensive Care Unit (ICU), 13% in Oncology and 10% in Gastroenterology. C. albicans was the most common species (32 cases=48%), followed by C. glabrata (17 cases=26%) and C. parapsilosis (12 cases=18%), a significant production of phospholipase in all strains of C. albicans was detected. Among Candida non-albicans species, the production of this enzyme only occurred in 1/12 strains of C. parapsilosis. The expression acid protease production was detected in 48% of C. albicans and no strains of Candida non-albicans. All species of Candida were susceptible to amphotericin B. The rate of susceptibility to fluconazole was 100% for albicans and C. parapsilosis. Decreased susceptibility to fluconazole was mostly seen with C. glabrata, which was 76.5% susceptible in a dose-dependent manner. The echinocandins showed a good performance for C. albicans, and maintained a good MIC distribution in C. glabrata.

Gardnerella, Trichomonas vaginalis, Candida, Chlamydia trachomatis, Mycoplasma hominis and Ureaplasma urealyticum in the genital discharge of symptomatic fertile and asymptomatic infertile women.

This study aimed to establish the different prevalence of the microorganisms investigated in the two groups considered: fertile women with symptoms and asymptomatic women with infertility problems. The data from women (n= 952) investigated for two years for quality of genital discharge and the presence of Gardnerella vaginalis, Trichomonas vaginalis, Candida species, Streptococcus agalactiae, Mycoplasma hominis, Ureaplasma urealyiticum and Chlamydia trachomatis were retrospectively analyzed. In the population of fertile women with symptoms the microrganisms most frequently involved are Gardnerella vaginalis (26.6%), Candida species (12.1%) and Streptococcus agalactiae (9.2%). The genital discharges of asymptomatic women with infertility problems are characterized by a prevalence of Gardnerella vaginalis (19.7%), Enterobacteriaceae or Enterococci (12.1%) and Streptococcus agalactiae (8.6%). The reduction of vaginal lactobacilli flora and the presence of an elevated number of polymorphonucleates in the vaginal discharge are important parameters to consider for the evaluation of the health status of the human female urogenital tract. Our results indicate that is important to culture the vaginal discharge for Streptococcus agalactiae and for prevalence of Enterobacteriaceae and Enterococci. Lastly, the reasons for the prevalence of some microorganisms (Gardnerella vaginalis, Enterobacteriaceae and Enterococci, Streptococcus agalactiae) in the population of infertile asymptomatic women need to be better analyzed especially after the recent studies correlating idiopathic infertility with the presence of cervical cytokines in women with an abnormal vaginal flora.

Aspergillus left ventricular assist device endocarditis.

Left ventricular assist device (LVAD) support is an established therapy for patients with end-stage heart failure as a bridge to transplant; its usage as an alternative for those patients not eligible for transplant is not an established therapy yet. A 68-year-old male had a Thoratec-Heartmate LVAD implanted as destination therapy. After an uneventful (apart from early fever) recovery in the intensive care unit, the patient developed an intractable high temperature, and generalized sepsis and died 21 days following implant. The white cell blood count never exceeded the guard limits, and the patient succumbed with severe LVAD valve malfunction. At post-mortem examination friable material consisting of fungal hyphae was found on the inflow and outflow valves. According to published clinical trials, infection accounts for more than 40% of mortality in LVAD supported patients. Fungal LVAD endocarditis is a particularly deadly disease. Successful management requires a high level of suspicion and timely institution of antifungal therapy to control the infection. This has led some authors to recommend empiric antifungal therapy in LVAD recipients with culture-negative sepsis unresponsive to broad-spectrum antibiotics.