Bacterial and fungal superinfections are detected at higher frequency in critically ill patients affected by SARS CoV-2 infection than negative patients and are associated to a worse outcome.

Patients with viral infections are at higher risk to acquire bacterial and fungal superinfections associated with a worse prognosis. We explored this critical point in the setting of patients with severe COVID-19 disease. The study included 1911 patients admitted to intensive care unit (ICU) during a 2-year study period (March 2020-March 2022). Of them, 713 (37.3%) were infected with SARS-CoV-2 and 1198 were negative (62.7%). Regression analysis was performed to determine risk factors associated with the presence of bacterial and/or fungal superinfections in SARS-CoV-2 patients and to evaluate predictors of ICU mortality. Of the 713 patients with SARS-CoV-2 infection, 473 (66.3%) had respiratory and/or bloodstream bacterial and/or fungal superinfections, while of the 1198 COVID-19-negative patients, only 369 (30%) showed respiratory and/or bloodstream bacterial and/or fungal superinfections (p < 0.0001). Baseline characteristics of COVID-19 patients included a median age of 66 (interquartile range [IQR], 58-73), a predominance of males (72.7%), and the presence of a BMI higher than 24 (median 26; IQR, 24.5-30.4). Seventy-four percent (527, 73.9%) had one or more comorbidities and 135 (18.9%) of them had received previous antibiotic therapy. Furthermore, most of them (473, 66.3%) exhibited severe radiological pictures and needed invasive mechanical ventilation. Multivariate logistic regression analysis showed that 1 unit increment in BMI rises the risk of bacterial and/or fungal superinfections acquisition by 3% and 1-day increment in ICU stays rises the risk of bacterial and/or fungal superinfections acquisition by 11%. Furthermore, 1-day increment in mechanical ventilation rises the risk of bacterial and/or fungal superinfection acquisition by 2.7 times. Furthermore, patients with both bacterial and fungal infections had a significantly higher mortality rate than patients without superinfections (45.8% vs. 26.2%, p < 0.0001). Therefore, bacterial and fungal superinfections are frequent in COVID-19 patients admitted to ICU and their presence is associated with a worse outcome. This is an important consideration for targeted therapies in critically ill SARS-CoV-2 infected patients to improve their clinical course.

First Italian report of a liver abscess and metastatic endogenous endophthalmitis caused by ST-23 hypervirulent Klebsiella pneumoniae in an immunocompetent individual.

BACKGROUND: Klebsiella pneumoniae is a common species in the gut of mammals and is widely distributed in the environment. However, the environmental source of hvKp that precedes gut colonization is unclear, but once that it reaches the gut there is a possible generalized spread y fecal-oral transmission especially in endemic areas. Liver abscess might develop when the bacteria, using its virulence factors, cross the intestinal barrier and invade the liver by the portal circulation. This syndrome, prevalent mostly in Asian countries, is increasingly reported in Western Countries and leaves open questions about the source of infection. CASE: Here we describe for the first time in Italy, a case of pyogenic liver abscess caused by a hypervirulent Klebsiella pneumoniae (HvKp) complicated by endophthalmitis and other metastatic infections in lung and prostate in an immunocompetent Chinese healthy individual with no recent travel in Asia. CONCLUSION: This case underlines the need for increased awareness of hypervirulent K. pneumoniae, even in settings where it occurs infrequently and where there are not evident epidemiological links.

Strongyloides stercoralis infection: an underlying cause of invasive bacterial infections of enteric origin. Results from a prospective cross-sectional study of a northern Italian tertiary hospital.

PURPOSE OF THE STUDY: We assessed the prevalence of S. stercoralis in a cohort of inpatients with invasive bacterial infections of enteric origin to investigate whether the parasite may facilitate these bacterial infections even in the absence of larval hyperproliferation. METHODS: We performed a prospective cross-sectional study in a hospital in northern Italy. Subjects admitted due to invasive bacterial infection of enteric origin and potential previous exposure to S. stercoralis were systematically enrolled over a period of 10 months. S. stercoralis infection was investigated with an in-house PCR on a single stool sample and with at least one serological method (in-house IFAT and/or ELISA Bordier). Univariate, bi-variate and logistic regression analyses were performed. RESULTS: Strongyloidiasis was diagnosed in 14/57 patients (24.6%; 95% confidence interval 14.1-37.8%) of which 10 were Italians (10/49, 20.4%) and 4 were migrants (4/8, 50.0%). Stool PCR was performed in 43/57 patients (75.4%) and no positive results were obtained. Strongyloidiasis was found to be significantly associated (p ≤ 0.05) with male gender, long international travels to areas at higher endemicity, deep extra-intestinal infectious localization and solid tumors. In the logistic regression model, increased risk remained for the variables deep extra-intestinal infectious localization and oncologic malignancy. CONCLUSIONS: Our findings suggest a new role of chronic strongyloidiasis in favoring invasive bacterial infections of enteric origin even in the absence of evident larval dissemination outside the intestinal lumen. Further well-designed studies should be conducted to confirm our results, and possibly establish the underlying mechanisms.

Streptococcus Pneumoniae Detection Long Time After Death in a Fatal Case of Waterhouse-Friderichsen Syndrome.

We report a fatal case of Waterhouse-Friderichsen syndrome in a 64-year-old man. The diagnosis, suspected during the autopsy (performed 63 hours after death), was confirmed through the successful detection of Streptococcus pneumoniae DNA and antigens in samples (blood and liver) collected during the autopsy. These results conformed with blood cultures performed antemortem, which became available only the day after the autopsy. The case underlines the need to collect biological material (liver and blood samples) during autopsy for microbiological investigations, although the collection is performed a long time after the death, suggesting that a liver sample works for DNA and liver and blood work for Streptococcus pneumoniae antigen detection.

In vitro activity of solithromycin against erythromycin-resistant Streptococcus agalactiae.

The in vitro antibacterial activity of solithromycin (CEM-101) against macrolide-resistant isolates (n=62) of Streptococcus agalactiae (group B streptococcus [GBS]) was determined. Phenotypic characterization of macrolide-resistant strains was performed by double-disc diffusion testing. A multiplex PCR was used to identify the erm(B), erm(TR), and mef(A/E) genes, capsular genotypes, and alpha-like (Alp) protein genes from the GBS strains. Determination of MIC was carried out using the microdilution broth method. The Etest method was used for penicillin, azithromycin, clarithromycin, and erythromycin. Solithromycin had a MIC50 of ≤0.008 µg/ml and a MIC90 of 0.015 µg/ml against macrolide-susceptible S. agalactiae. These MICs were lower than those displayed by penicillin (MIC50 of 0.032 µg/ml and MIC90 of 0.047 µg/ml), the antibiotic agent of choice for prophylaxis and treatment of GBS infections. Against macrolide-resistant S. agalactiae, solithromycin had a MIC50 of 0.03 µg/ml and a MIC90 of 0.125 µg/ml. Against erm(B) strains, solithromycin had a MIC50 of 0.03 µg/ml and a MIC90 of 0.06 µg/ml, while against mef(A) strains, it had a MIC50 of 0.03 µg/ml and a MIC90 of 0.125 µg/ml. Most erythromycin-resistant GBS strains were of serotype V (64.5%) and associated significantly with alp2-3. Moreover, a statistically significant association was observed between the constitutive macrolide-lincosamide-streptogramin B resistance (cMLSB) phenotype and the erm(B) gene-carrying strains, the alp2-3 gene and the M phenotype, and the mef(A/E) gene and epsilon. Overall, our results show that solithromycin had lower or similar MICs than penicillin and potent activity against macrolide-resistant strains independent of their genotype or phenotype, representing a valid therapeutic alternative where ß-lactams cannot be used.

Nosocomial outbreak of the pandemic Influenza A (H1N1) 2009 in critical hematologic patients during seasonal influenza 2010-2011: detection of oseltamivir resistant variant viruses.

BACKGROUND: The pandemic influenza A (H1N1) 2009 (H1N1pdm09) virus infection caused illness and death among people worldwide, particularly in hematologic/oncologic patients because influenza infected individuals can shed virus for prolonged periods, thus increasing the chances for the development of drug-resistant strains such as oseltamivir-resistant (OST-r) variant. METHODS: The aim of our study was to retrospectively evaluate the clinical importance of OST-r variant in circulating strains of the pandemic H1N1pdm09 virus. By means of RT-PCR and Sanger sequencing we analysed the presence of OST-r variant in 76 H1N1pdm09 laboratory-confirmed cases, hospitalized at the hematologic/oncologic ward at Spedali Civili of Brescia -Italy. RESULTS: Out of 76 hospitalized hematologic/oncologic patients, 23 patients (30.2%) were infected by H1N1pdm09 virus. Further investigation revealed that 3 patients were positive for the OST-r variant carrying the H275Y mutation. All the 23 infected patients were immuno-compromised, and were under treatment or had been treated previously with oseltamivir. Three patients died (13%) after admission to intensive care unit and only one of them developed H275Y mutation. CONCLUSIONS: Our retrospective observational study shows that pandemic influenza A (H1N1) 2009 virus can cause significant morbidity and even mortality in hematologic/oncologic patients and confirms the high rate of nosocomial transmission of pandemic H1N1pdm09 virus in these critical subjects. Indeed, the reduction in host defences in these hospitalized patients favoured the prolonged use of antiviral therapy and permitted the development of OST-r strain. Strategies as diagnostic vigilance, early isolation of patients and seasonal influenza A(H1N1) vaccination may prevent transmission of influenza in high risk individuals.

In Vitro Activity of Cefiderocol on Multiresistant Bacterial Strains and Genomic Analysis of Two Cefiderocol Resistant Strains.

Cefiderocol is a new siderophore cephalosporin that is effective against multidrug-resistant Gram-negative bacteria, including carbapenem-resistant strains. The aim of this study was to evaluate the activity of this new antimicrobial agent against a collection of pathogens using broth microdilution assays and to analyze the possible mechanism of cefiderocol resistance in two resistant Klebsiella pneumoniae isolates. One hundred and ten isolates were tested, comprising 67 Enterobacterales, two Acinetobacter baumannii, one Achromobacter xylosoxidans, 33 Pseudomonas aeruginosa and seven Stenotrophomonas maltophilia. Cefiderocol showed good in vitro activity, with an MIC < 2 µg/mL, and was able to inhibit 94% of the tested isolates. We observed a resistance rate of 6%. The resistant isolates consisted of six Klebsiella pneumoniae and one Escherichia coli, leading to a resistance rate of 10.4% among the Enterobacterales. Whole-genome sequencing analysis was performed on two cefiderocol-resistant Klebsiella pneumoniae isolates to investigate the possible mutations responsible for the observed resistance. Both strains belonged to ST383 and harbored different resistant and virulence genes. The analysis of genes involved in iron uptake and transport showed the presence of different mutations located in fhuA, fepA, iutA, cirA, sitC, apbC, fepG, fepC, fetB, yicI, yicJ, and yicL. Furthermore, for the first time, to the best of our knowledge, we described two Klebsiella pneumoniae isolates that synthesize a truncated fecA protein due to the transition from G to A, leading to a premature stop codon in the amino acid position 569, and a TonB protein carrying a 4-amino acid insertion (PKPK) after Lysine 103. In conclusion, our data show that cefiderocol is an effective drug against multidrug-resistant Gram-negative bacteria. However, the higher resistance rate observed in Enterobacterales underlines the need for active surveillance to limit the spread of these pathogens and to avoid the risks associated with the emergence of resistance to new drugs.

Ex-vivo study about antimicrobial effectiveness of phytic acid against Enterococcus faecalis into root canals.

BACKGROUND: Endodontic failure is due to the persistence of microorganisms, especially Enterococcus faecalis, which have become resistant to disinfection measures. Sodium hypochlorite has been traditionally used, whereas phytic acid, a natural irrigant, needs to be further investigated. METHODS: This ex-vivo study compared the antibacterial effectiveness of 0.9% saline solution, 5% sodium hypochlorite and 5% phytic acid against Enterococcus faecalis ATCC 29212 into the apical root canal third of 96 single-rooted extracted teeth, after 1-minute push-and-pull irrigation - a basic irrigation technique. Survived microorganisms were evaluated both through a traditional colony-forming-unit count and introducing the viability PCR, which precisely detects only DNA from intact cells: dead and damaged cells were excluded thanks to a propidium monoazide dye. RESULTS: The culture methods showed that 5% sodium hypochlorite (median: 0 CFU/mL) has a significant greater antibacterial effectiveness (P<0.001) compared both to 0.9% saline solution (median: 4.76 CFU/mL) and 5% phytic acid (median: 0.25 CFU/mL). However, 5% phytic acid proved to be significantly more effective (P<0.001) than 0.9% saline solution. The viability PCR did not reveal a significant difference between 5% sodium hypochlorite (median: 4.12×104 survived bacteria) and 0.9% saline solution (median: 8.45×104 survived bacteria). Five percent phytic acid (median: 0.83×104 survived bacteria) was significantly more effective than both 0.9% saline solution (P<0.001) and 5% sodium hypochlorite (P<0.001). CONCLUSIONS: The findings suggest that 5% phytic acid works against root canal bacteria even with a basic irrigation technique and viability PCR seems to lead to more reliable and sensitive data rather than the culture methods.

What empiric treatment should be given for suspected bacterial keratitis in Northern Italy?

There is a lack of epidemiological studies reporting bacteria profiles, susceptibility, and suggested empiric (first line) treatment in Northern Italy. Our internal audit of corneal scraping for microbial keratitis at University of Brescia reports 116 bacterial strains isolated between January 1, 2013, and December 31, 2020. All cases had at least an epithelial defect of 1 mm in diameter. 36.2% (42) were Gram-positive, while 63.7% (74) Gram negative. In our results Gram-negatives are sensitive to ciprofloxacin in 94.5% and Pseudomonas in 95%. Grampositives are sensitive to teicoplanin in 91,1%. Those data may help to establish empiric treatment in case of bacterial keratitis.

Management of Invasive Infections due to a Rare Arthroconidial Yeast, Saprochaete capitata, in Two Patients with Acute Hematological Malignancies.

Saprochaete capitata is an arthroconidial yeast, found principally in the environment, even if it belongs also to the normal microbial flora that colonize human subjects. This yeast is increasingly associated with invasive infections in hematological patients, in particular in those affected by acute leukemia. An important risk factor that predisposes to this infection is the profound neutropenia present in such immunocompromised patients. Saprochaete spp. were found resistant to both echinocandins and fluconazole so the treatment is often difficult. Here, we report two cases of sepsis in two patients with acute leukemia. All of them had fatal events, due to the worsening of their clinical condition. An early diagnosis and appropriate management of these pathogens is important in consideration of the poor prognosis associated to these fungal invasive infections.

Detection of microbial contamination in dialysis water and gastrointestinal endoscopes by the Uro4 HB&L™ system.

Patients undergoing haemodialysis are exposed to a great volume of dialysis water and therefore its microbial analysis is important for patient safety. Furthermore, microbial surveillance is reported in several guidelines as a necessary means to identify contamination of gastrointestinal endoscopes in order to reduce the potential of infection risk. Here we evaluated the Uro4 HB&L™ automated system to detect microbial contamination in dialysis water and gastrointestinal endoscopes. A total of 222 samples were collected during a six month period. Dialysis water and gastrointestinal endoscopes were evaluated both with the reference culture methods and the Uro4 HB<M system. Bacterial identification was performed using a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The results show that the Uro4 HB<M system has high specificity but a lesser sensitivity than traditional culture method, even if it might allow the identification of more exigent bacteria in terms of nutrition. The Uro4 HB<M system gives positive results in less time than culture method but the possibility to generate false negative results imposes that it should be associated with a traditional 48h agar culture.

Extensively drug-resistant Acinetobacter baumannii isolated from intensive care units in northern Italy: a genomic approach to characterize new sequence types.

Aim: This study aims to characterize clinical strains of Acinetobacter baumannii with an extensively drug-resistant phenotype. Methods: VITEK® 2, Etest® method and broth microdilution method for colistin were used. PCR analysis and multilocus sequence typing Pasteur scheme were performed to identify bla-OXA genes and genetic relatedness, respectively. Whole-genome sequencing analysis was used to characterize three isolates. Results: All the isolates were susceptible only to polymyxins. blaOXA-23-like gene was the only acquired carbapenemase gene in 88.2% of the isolates. Multilocus sequence typing identified various sequence types: ST2, ST19, ST195, ST577 and ST632. Two new sequence types, namely, ST1279 and ST1280, were detected by whole-genome sequencing. Conclusion: This study showed that carbapenem-resistant A. baumannii isolates causing infections in intensive care units almost exclusively produce OXA-23, underlining their frequent spread in Italy.

Detection and molecular characterization of enteric viruses in children with acute gastroenteritis in Northern Italy.

Enteric viral infections are a major concern for public health, and viral acute gastroenteritis is the principal cause of pediatric morbidity and mortality worldwide, mostly in developing countries. The purpose of this study was to determine the prevalence of different enteric viruses detected in a pediatric population with acute gastroenteritis symptoms, and to characterize the strains detected. Stools were collected from children, aged from 2 months to 15 years old, admitted to one of the main hospitals of Northern Italy, between November 2015 and October 2016. Stools were tested for nine enteric viruses (adenovirus, rotavirus A, norovirus, astrovirus, sapovirus, enterovirus, parechovirus, bocavirus and aichivirus) by molecular methods. Furthermore, rotavirus, norovirus and adenovirus were deeply characterized by nucleotide sequencing and phylogenetic analysis. A total of 151 out of 510 (29.6%) stools analyzed resulted positive for at least one of the enteric virus investigated. The most common virus detected was rotavirus A (53/151, 35.1%), followed by norovirus (39/151, 25.8%), adenovirus (35/151, 23.1%), sapovirus (9/151, 6%), enterovirus (5/151, 3.3%), astrovirus (5/151, 3.3%), parechovirus (4/151, 2.6%) and bocavirus (1/151, 0.6%). Aichi virus was not detected in any sample. Co-infections were detected in 12 out of 510 faecal samples (2.3%). These data improved the knowledge of the enteric viruses circulating in children in Northern Italy. In fact, besides rotavirus, adenovirus and norovirus, several viruses circulated across the whole year in the pediatric population object of this study. The introduction of specific viral diagnosis in our clinical setting will improve patient care by reducing unnecessary use of antibiotics addressing the right etiologic diagnosis.

Myroides odoratimimus urinary tract infection in an immunocompromised patient: an emerging multidrug-resistant micro-organism.

Background: Myroides spp. are common environmental organisms and they can be isolated predominantly in water, soil, food and in sewage treatment plants. In the last two decades, an increasing number of infections such as urinary tract infections and skin and soft tissue infections, caused by these microorganisms has been reported. Selection of appropriate antibiotic therapy to treat the infections caused by Myroides spp. is difficult due to the production of a biofilm and the organism's intrinsic resistance to many antibiotic classes. Case presentation: We report the case of a 69-year-old immunocompromised patient who presented with repeated episodes of macroscopic haematuria, from Northern Italy.A midstream urine sample cultured a Gram negative rod in significant amounts (> 105 colony-forming units (cfu)/mL), which was identified as Myroides odoratimimus. The patient was successfully treated with trimethoprim/sulfamethoxazole after antibiotic susceptibility testing confirmed its activity. Conclusion: This case underlines the emergence of multidrug resistant Myroides spp. which are ubiquitous in the environment and it demands that clinicians should be more mindful about the role played by atypical pathogens, which may harbour or express multidrug resistant characteristics, in immunocompromised patients or where there is a failure of empiric antimicrobial therapy.

Fulminant septic shock caused by Capnocytophaga canimorsus in Italy: Case report.

Capnocytophaga canimorsus infection was recently recognized as a zoonosis. We report the first case of fulminant septic shock in Italy caused by this pathogen. The patient, with a history of splenectomy, died at the main hospital in Brescia with a presumptive diagnosis of sepsis. PCR and sequencing on post mortem samples confirmed C. canimorsus as a causative organism. Our purpose is to alert medical professionals to the virulence of C. canimorsus in asplenic and immunocompromised patients.

Microbiological characterization and effect of resin composites in cervical lesions.

BACKGROUND: Non carious cervical lesions associated to muscle hyperfunctions are increasing. Microhybrid resin composites are used to restore cervical abfractions. The purpose of this study was to investigate if resin composites modify tooth plaque, inducing an increment of cariogenic microflora and evaluate their effect, in vivo and in vitro, against S. mutans. MATERIAL AND METHODS: Eight abfractions were restored with two microhybrid resin composites (Venus, Heraeus-Kulzer® and Esthet-X, Dentsply®), after gnatological therapy, in three patients with muscle hyperfunctions. For each abfraction three samples of plaque were taken from the cervical perimeter: before the restoration, one week and three months after restoration. The samples were evaluated both by traditional microbiological methods and by Polymerase Chain Reaction (PCR). In vitro, disk-shaped specimens of the two composites were prepared to estimate the effects against pre-cultured S. mutans, after incubation at 37°C for 24h and assessed by a turbidimetric technique. RESULTS: In vivo no differences were found in plaque growth, for all samples, before and after restoration with both composites; in vitro, instead, a significant reduction of S. mutans growth was found between specimens of two composites (Mann-Whitney U-test p>0,06). CONCLUSIONS: In this study a relevant consideration was elicited: composite materials, in vivo, do not modify plaque composition of non carious cervical lesions to a potential cariogenic plaque. Key words:Abfraction, restoration, S. mutans, composite, class V.

Analysis of Point Mutations in the pbp2x, pbp2b, and pbp1a Genes of Streptococcus agalactiae and Their Relation with a Reduced Susceptibility to Cephalosporins.

The emergence of group B Streptococcus (GBS) isolates with reduced penicillin susceptibility (PRGBS) and their tendency to be nonsusceptible to fluoroquinolones prompted us to analyze the possible presence of amino acid mutations in penicillin-binding proteins (PBPs) (PBP2X, PBP1A, and PBP2B) from a collection of fluoroquinolone-resistant GBS isolates. We analyzed 21 GBS isolates resistant to levofloxacin. Sequence analysis of genes for PBPs was performed. The minimal inhibitory concentrations (MICs) for penicillin, ceftibuten, cefaclor, cefixime, cefotaxime, and ceftizoxime were performed by the Etest method and by broth microdilution method. The isolates were furthermore characterized by PCR-based capsular typing and analysis of surface protein genes. Genetic relatedness among the isolates was examined by multilocus sequence typing. Phylogenetic analysis of PBPs sequences was performed by Molecular Evolutionary Genetics Analysis software (MEGA7). All isolates were susceptible to penicillin, even if different mutations were detected in all PBPs in most of the isolates (12/21, 57%). However, we observed a reduced susceptibility to cefixime in seven isolates and to cefaclor in six isolates. These PSGBS isolates shared an I377V mutation in PBP2X and a T145A mutation in PBP1A. Most of the isolates belongs to the clonal complex 1, has serotype III and rib as surface protein. The results of phylogenetic comparative analysis show that several genetic lineages of our isolates with reduced susceptibility to cefixime/cefaclor have been independently emerging through the accumulation of mutations in their pbp genes, especially in pbp1a. If the MICs of penicillins and cephalosporins for GBS increase, careful epidemiological surveillance on this issue is recommended.

Analysis of mutations in DNA gyrase and topoisomerase IV of Ureaplasma urealyticum and Ureaplasma parvum serovars resistant to fluoroquinolones.

This study aims to determine the prevalence of fluoroquinolone resistance of Ureaplasma biovars and serovars isolated from urogenital clinical samples and determine the underlying molecular mechanism for quinolone resistance for all resistant isolates. Of 105 samples confirmed as positive for U. urealyticum/U. parvum, 85 were resistant to quinolones by the Mycoplasma-IST2 kit. However, only 43 out of 85 quinolone resistant isolates had amino acid substitutions in GyrA, GyrB, ParC and ParE proteins underlining that this assay have mis-identified as fluoroquinolone resistant 42 isolates. The known ParC E87K and ParC S83L mutations were found in 1 and 10 isolates, respectively. An original mutation of ureaplasmal ParC (E87Q, 1 isolate) was found. Furthermore, we found a ParE R448K mutation in one isolate, already described. Among the additional alterations detected, the most prevalent mutation found was L176F in GyrA protein in 18 isolates with single infection and in 3 isolates with mixed ureaplasma infections. Mutations in GyrB (E502Q, 4 isolates), ParE (Q412K, Q412P, Q412T, 3 independent isolates), whose role is unknown, were also found. Other sporadic mutations in the four genes were identified. This investigation is the result of monitoring the data for molecular fluoroquinone resistance in Ureaplasma spp. in Italy. Resulting that this acquired resistance is high and that continued local epidemiological studies are essential to monitor and document their antimicrobial resistance trends.