Diagnostic and infection control strategies for Clostridioides difficile infections in a setting of high antimicrobial resistance prevalence.

Clostridioides difficile (CD) is a major nosocomial pathogen and the leading cause of antibiotic-associated diarrhoea. In light of the strong association between antimicrobial use and CD infections (CDI), it may be hypothesised that areas at higher prevalence of antimicrobial resistance, like the region of Campania in southern Italy, could also have a higher rate of CDI. In this multicentre, region-based, prospective study, we analysed such issues, exploiting CDI incidence data collected from local hospitals. In 2016, the Italian National Centre for Disease Control supported a project involving three Italian regions: Friuli Venezia Giulia, Lazio and Campania. In Campania, a network of 49 hospitals willing to participate in the project was created. The project consisted of two phases: a survey on practice patterns concerning CDI and an epidemiological surveillance study. We identified a stringent need to improve awareness about CDI among the regional health-care community, as a widespread lack of surveillance programmes for CDI control was observed (existing in only 40% of participating facilities). Moreover, almost half of the participating hospitals (n=16, 43%) had no standardised procedures or protocols to control and prevent CDI. In the second phase of the study, we collected data of CDI cases during a six-month surveillance programme. In all, 87 CDI cases were observed, for a total of 903,334 patient bed-days and 122,988 admissions. According to the above data, CDI incidence was 0.96 cases/10000 patient bed-days, much lower than expected based on prior studies conducted elsewhere. The results of our study suggest CDI remains a rather neglected clinical issue in Campania. Despite a high burden of antimicrobial resistance and antimicrobial use in our geographic setting, we observed a very low incidence of CDI. Such a low incidence could be explained by underdiagnosis, but could also be related to actual diet, the lower patient age or the specific genetic background. However, further studies are warranted to either confirm or rebut the above hypotheses.

Molecular Epidemiology and Virulence Profiles of Colistin-Resistant Klebsiella pneumoniae Blood Isolates From the Hospital Agency "Ospedale dei Colli," Naples, Italy.

Resistance to colistin is increasingly reported in Klebsiella pneumoniae clinical isolates. The aim of this study was to analyze the molecular epidemiology and virulence profiles of 25 colistin-resistant K. pneumoniae blood isolates from the Hospital Agency "Ospedale dei Colli," Naples, Italy, during 2015 and 2016. Colistin MIC values of isolates ranged from 4 to 256 mg/L. The inactivation of the mgrB gene, encoding a negative regulator of the PhoQ/PhoP signaling system, was the most frequent mechanism of colistin resistance found in 22 out of 25 isolates. Of these, 10 isolates assigned to ST512 and PFGE types A and A4 showed identical frameshift mutation and premature termination of mgrB gene; 4 isolates assigned to ST258 and PFGE types A1 showed non-sense, frameshift mutation, and premature termination; 3 and 1 isolates assigned to ST258 and PFGE A2 and ST512 and PFGE A3, respectively, had insertional inactivation of mgrB gene due to IS5-like mobile element; 2 isolates assigned to ST101 and 1 to ST392 had missense mutations in the mgrB gene, 1 isolate assigned to ST45 showed insertional inactivation of mgrB gene due to IS903-like mobile element. phoQ missense mutations were found in 2 isolates assigned to ST629 and ST101, respectively, which also showed a missense mutation in pmrA gene. The mcr-1-2-3-4 genes were not detected in any isolate. Colistin-resistant K. pneumoniae isolates showed variable virulence profiles in Galleria mellonella infection assays, with the infectivity of two isolates assigned to ST45 and ST629 being significantly higher than that of all other strains (P < 0.001). Interestingly, colistin MIC values proved to make a significant contribution at predicting lethal doses values (LD50 and LD90) of studied isolates in G. mellonella. Our data show that MgrB inactivation is a common mechanism of colistin resistance among K. pneumoniae in our clinical setting. The presence of identical mutations/insertions in isolates of the same ST and PFGE profile suggests the occurrence of clonal expansion and cross-transmission. Although virulence profiles differ among isolates irrespective of their genotypes, our results suggest that high colistin MIC could predict lower infectivity capability of the isolates.

A Novel IncA/C1 Group Conjugative Plasmid, Encoding VIM-1 Metallo-Beta-Lactamase, Mediates the Acquisition of Carbapenem Resistance in ST104 Klebsiella pneumoniae Isolates from Neonates in the Intensive Care Unit of V. Monaldi Hospital in Naples.

The emergence of carbapenemase producing Enterobacteriaceae has raised major public health concern. The aim of this study was to investigate the molecular epidemiology and the mechanism of carbapenem resistance acquisition of multidrug-resistant Klebsiella pneumoniae isolates from 20 neonates in the neonatal intensive care unit (NICU) of the V. Monaldi Hospital in Naples, Italy, from April 2015 to March 2016. Genotype analysis by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) identified PFGE type A and subtypes A1 and A2 in 17, 2, and 1 isolates, respectively, and assigned all isolates to sequence type (ST) 104. K. pneumoniae isolates were resistant to all classes of ß-lactams including carbapenems, fosfomycin, gentamicin, and trimethoprim-sulfamethoxazole, but susceptible to quinolones, amikacin, and colistin. Conjugation experiments demonstrated that resistance to third-generation cephems and imipenem could be transferred along with an IncA/C plasmid containing the extended spectrum ß-lactamase blaSHV -12 and carbapenem-hydrolyzing metallo-ß-lactamase blaV IM-1 genes. The plasmid that we called pIncAC_KP4898 was 156,252 bp in size and included a typical IncA/C backbone, which was assigned to ST12 and core genome (cg) ST12.1 using the IncA/C plasmid MLST (PMLST) scheme. pIncAC_KP4898 showed a mosaic structure with blaV IM-1 into a class I integron, blaSHV -12 flanked by IS6 elements, a mercury resistance and a macrolide 2'-phosphotransferase clusters, ant(3″), aph(3″), aacA4, qnrA1, sul1, and dfrA14 conferring resistance to aminoglycosides, quinolones, sulfonamides, and trimethoprim, respectively, several genes predicted to encode transfer functions and proteins involved in DNA transposition. The acquisition of pIncAC_KP4898 carrying blaV IM-1 and blaSHV -12 contributed to the spread of ST104 K. pneumoniae in the NICU of V. Monaldi Hospital in Naples.

Surveillance of healthcare-associated infections in a neonatal intensive care unit in Italy during 2006-2010.

BACKGROUND: Healthcare-associated infections (HAIs) are a frequent complication associated with hospitalization of infants in neonatal intensive care units (NICUs). The aim of this study was to evaluate and describe the results of surveillance of HAIs in a III level NICU in Naples, Italy during 2006-2010. METHODS: The surveillance covered 1,699 neonates of all birth weight (BW) classes with >2 days NICU stay. Infections were defined using standard Centers for Disease Control and Prevention definitions adapted to neonatal pathology and were considered to be healthcare-associated if they developed >2 days after NICU admission. RESULTS: One hundred-fifty-three HAIs were diagnosed with a frequency of 9% and an incidence density of 3.5 per 1000 days of hospital stay. HAIs developed in all BW classes, but patients weighing≤1000 g at birth were more affected with a decreasing trend from the lowest to the highest BW classes. Sepsis proved to be the most frequent infection (44.4%), followed by urinary tract infection (UTI) (28.8%), pneumonia (25.5%) and meningitis (1.3%). Device associated infections (i.e. central line-associated bloodstream infections (BSIs), umbilical catheter-associated BSI and ventilator associated pneumonias (VAPs) represented 64.1% of all HAIs. Most frequent pathogens responsible for all types of infections were: P. aeruginosa (17%), C. parapsilosis (16.3%), E. coli (13.1%), C. albicans (10.5%), non- extended spectrum beta-lactamase (ESBL) K. pneumoniae (7.8%), and coagulase-negative Staphylococci (5.2%). No microbiological diagnosis was achieved for 6.5% of infections. CONCLUSIONS: HAIs developed in all BW classes but low BW neonates were at major risk to acquire HAIs in our NICU. Use of central line-, umbilical-catheter and mechanical ventilation was associated with higher risk of infection. Our findings highlight the importance of an extensive surveillance approach in the NICU setting, which includes all BW classes of neonates and monitors infections associated with the use of medical devices.

[Experimental prospective cognitive study on the evaluation of connection of fatigue perception and depression among caregivers and team treating cancer patients receiving oral chemotherapy to systemic chemotherapy. Equi-Car 10 study]. / Studio sperimentale prospettico conoscitivo di valutazione della relazione della percezione di fatigue e depressione tra caregiver, equipe curante e pazienti oncologici in trattamento chemioterapico con terapie somministrate per via orale verso terapie somministrate per via sistemica Studio Equipe-Caregivers 10 (Studio Equi-Car 10).

OBJECTIVE: The Equi-Car 10 is an spontaneous, observational study. The objective was to assess the connection of fatigue perception and depression of caregivers, oncological team and oncological patients in oral chemotherapy to intravenous METHOD: The study recruited 60 patients and 60 caregivers. The study was carried out on patients with lung, breast and colon cancer requiring chemotherapy, and family members were chosen as caregivers. Patients, caregivers and doctors filled in FACT-F and Zung questionnaires at the opening day of the medical record, after three and six cycles of chemotherapy, and three months after the end of the treatment. RESULTS: The Zung depression and Functional Assessment of Cancer therapy-Fatigue (FACT-F) showed: (a) on patients treated with intravenous therapy showed significantly higher levels of fatigue and depression than those of patients treated with oral therapy, even when the latter were treated for advanced disease; (b) on caregivers of patients treated with systemically administered therapies showed elevated levels of Fatigue and depression which proved to be significantly higher than those of caregivers of patients treated with oral therapies, even when treated for advanced disease; (c) on three oncologist has highlighted low levels of Fatigue and depression, for both types of patients, higher for patients with systemic therapy related to the management of side effects. CONCLUSIONS: The results showed that cancer patients and caregivers have high levels of fatigue and depression both related to the disease stage and to the mode of drug administration. It is necessary to provide cancer patients and caregivers with appropriate psychological support and preventive programs for secondary and relapse prevention.

Molecular epidemiology of multidrug-resistant Acinetobacter baumannii in a tertiary care hospital in Naples, Italy, shows the emergence of a novel epidemic clone.

The molecular epidemiology of multidrug-resistant Acinetobacter baumannii was investigated in two intensive care units of the V. Monaldi university hospital in Naples, Italy, from May 2006 to December 2007. Genotype analysis by pulsed-field gel electrophoresis (PFGE), trilocus sequence-based typing (3LST), and multilocus sequence typing (MLST) of A. baumannii isolates from 71 patients identified two distinct genotypes, one assigned to PFGE group A, 3LST group 1, and ST2 in 14 patients and the other to PFGE group B, 3LST group 6, and ST78 in 71 patients, that we named ST2/A and ST78/B, respectively. Of these, ST2/A corresponded to European clone II identified in the same hospital during 2003 and 2004; ST78/B was a novel genotype that was isolated for the first time in May 2006 but became prevalent during 2007. The ST78/B profile was also identified in five patients from two additional hospitals in Naples during 2007. The ST2/A and ST78/B isolates were resistant to all antimicrobials tested, including carbapenems, but were susceptible to colistin. Both ST2/A and ST78/B isolates possessed a plasmid-borne carbapenem-hydrolyzing oxacillinase gene, bla(OXA-58), flanked by ISAba2 and ISAba3 elements at the 5' and 3' ends, respectively. The selection of the novel ST78/B A. baumannii clone might have been favored by the acquisition of the bla(OXA-58) gene.

Pseudomonas aeruginosa in a neonatal intensive care unit: molecular epidemiology and infection control measures.

BACKGROUND: Pseudomonas aeruginosa, a non-fermentative, gram-negative rod, is responsible for a wide variety of clinical syndromes in NICU patients, including sepsis, pneumonia, meningitis, diarrhea, conjunctivitis and skin infections. An increased number of infections and colonisations by P. aeruginosa has been observed in the neonatal intensive care unit (NICU) of our university hospital between 2005 and 2007. METHODS: Hand disinfection compliance before and after an educational programme on hand hygiene was evaluated. Identification of microrganisms was performed using conventional methods. Antibiotic susceptibility was evaluated by MIC microdilution. Genotyping was performed by PFGE analysis. RESULTS: The molecular epidemiology of Pseudomonas aeruginosa in the NICU of the Federico II University hospital (Naples, Italy) and the infection control measures adopted to stop the spreading of P. aeruginosa in the ward were described. From July 2005 to June 2007, P. aeruginosa was isolated from 135 neonates and caused severe infections in 11 of them. Macrorestriction analysis of clinical isolates from 90 neonates identified 20 distinct genotypes, one major PFGE type (A) being isolated from 48 patients and responsible for 4 infections in 4 of them, four other distinct recurrent genotypes being isolated in 6 to 4 patients. Seven environmental strains were isolated from the hand of a nurse and from three sinks on two occasions, two of these showing PFGE profiles A and G identical to two clinical isolates responsible for infection. The successful control of the outbreak was achieved through implementation of active surveillance of healthcare-associated infections in the ward together with environmental microbiological sampling and an intense educational programme on hand disinfection among the staff members. CONCLUSION: P. aeruginosa infections in the NICU were caused by the cross-transmission of an epidemic clone in 4 neonates, and by the selection of sporadic clones in 7 others. An infection control programme that included active surveillance and strict adherence to hand disinfection policies was effective in controlling NICU-acquired infections and colonisations caused by P. aeruginosa.

Neonatal hyperbilirubinemia increases intestinal protein permeability and the prevalence of cow's milk protein intolerance.

AIMS: Bilirubin is a newly discovered modulator of the gut barrier in vitro and in vivo. We studied the effect of bilirubin on the serosal to mucosal intestinal permeability in vivo. We also investigated the prevalence of cow's milk protein intolerance (CMPI) in infants with moderate hyperbilirubinemia versus matched controls. METHODS: Faecal alpha 1 antitrypsin (a1AT) was used to monitor intestinal protein loss; a large cohort was prospectively followed for 12 months for sign and symptoms of CMPI. RESULTS: Neonates with hyperbilirubinemia had higher stool excretion of a1AT than controls (0.68 +/- 0.28 mg/g vs. 0.25 +/- 0.11 mg/g; p < 0.01). Faecal a1AT correlates with total serum bilirubin (TSB) (r = 0.85; p < 0.01). Also, in the first 12 months of life, formerly hyperbilirubinemic infants had an higher prevalence of CMPI (14/353 vs. 4/339; chi2= 4.018, p = 0.045). CONCLUSIONS: Neonatal hyperbilirubinemia increases stool protein loss and is also a mild risk factor for CMPI.

Unconjugated bilirubin modulates the intestinal epithelial barrier function in a human-derived in vitro model.

Unconjugated bilirubin promotes intestinal secretion without affecting nutrient digestion or absorption. In the current study, the effects of unconjugated bilirubin (UCB) on the barrier function of the intestinal epithelium were investigated. The apical side of human intestinal cell line Caco-2 monolayers was challenged with purified UCB. Transepithelial electrical resistance and paracellular fluxes of 10 kD Cascade blue conjugate dextran were measured. Cell monolayer viability was studied using LDH release and trypan blue exclusion tests. Redistribution of enterocyte tight junction occludin was studied by confocal microscopy. Bilirubin induced a dose-dependent decrease of transepithelial electrical resistance (TEER). This effect was maximal at 6 h and tended to be reversed at 48 h. Oxidated bilirubin was ineffective. Bilirubin significantly increased fluorescent dextran paracellular passage. Cell viability was not affected by UCB over the 5-200 nmol/L concentration range. Finally, bilirubin triggered a reversible redistribution of tight junctional occludin. UCB increases the permeability of intestinal epithelium. This effect is reversible, dependent on the redox status of the molecule and the rearrangement of the tight junction. These data attribute to bilirubin a novel role of functional modulator of intestinal paracellular permeability in vitro.

Molecular epidemiology of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in a neonatal intensive care unit.

OBJECTIVES: To investigate the molecular epidemiology of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae in the neonatal intensive care unit of a university hospital in Italy. METHODS: Antibiotic susceptibility was evaluated by disc diffusion and Etest. ESBLs were identified by isoelectric focusing, PCR and DNA sequencing analysis. Genotyping was performed by PFGE analysis. Conjugation was performed by broth mating. RESULTS: Molecular typing of K. pneumoniae isolates identified three distinct PFGE patterns. Isolates of PFGE profile A were isolated during an epidemic in 1996, while isolates of PFGE profiles B and C were sequentially isolated from September 2002 to December 2004, when 233 colonizations and 19 infections by K. pneumoniae occurred. All K. pneumoniae strains of different PFGE types were identified as ESBL producers. DNA sequencing of amplified beta-lactamase genes identified a novel bla(TEM) ESBL (bla(TEM-136)) along with bla(SHV-1) in chromosomal and plasmid DNA from K. pneumoniae of PFGE type A, respectively, and bla(TEM-1) and bla(SHV-12) in plasmid DNA from K. pneumoniae of PFGE types B and C. Conjugation experiments demonstrated that resistance to third-generation cephalosporins, along with an approximately 80 kb plasmid containing bla(SHV-12) and bla(TEM-1), was transferred from K. pneumoniae epidemic strains of PFGE types B and C to a susceptible Escherichia coli host at a frequency of 4 x 10(-6) and 1 x 10(-6) cfu/recipient cell, respectively. CONCLUSIONS: The selection of ESBL-producing clones and the transfer of the bla(SHV-12) ESBL gene between different clones were responsible for the spread of K. pneumoniae in the neonatal intensive care unit.

Early cord clamping protects at-risk neonates from polycythemia.

UNLABELLED: Neonatal polycythemia is a potentially lethal, multi-organ disease. We have performed a prospective, open-label study to test the hypothesis that an early cord clamping proximally to the neonate's abdomen could avert from the neonatal circulation a blood volume critical to the occurrence of polycythemia in at-risk neonates. Newborns were divided into group 1 (clamping time within 10 s) and group 2 (clamping time 11-120 s). Group 1 had statistically significant more blood volume sequestered in the cord and less manifestations of polycythemia. CONCLUSION: An early cord clamping is an effective and zero-cost way to prevent polycythemia in at-risk neonates.

Moderate hyperbilirubinemia induces a transient alteration of neonatal behavior.

OBJECTIVE: To investigate the behavioral changes induced by moderate hyperbilirubinemia in the otherwise healthy, untreated newborn infant. METHODS: Fifty term neonates (23 boys) with untreated moderate hyperbilirubinemia (median: 14.3 mg/dL; range: 13.2-20 mg/dL) and 50 matched control subjects with lower bilirubin concentrations (median: 9.1 mg/dL; range: 5.3-12 mg/dL) were administered the Brazelton Neonatal Behavioral Scale at 87 hours of life (range: 72-110 hours). A subgroup analysis was also performed at 104 hours of life (range: 96-134 hours) and at 3 weeks of age. RESULTS: At the first examination, all behavioral clusters were significantly altered in the group with moderate hyperbilirubinemia. The visual and auditory capabilities of the hyperbilirubinemic infant were especially compromised. Although social-interactive cluster scores significantly correlated both with serum bilirubinemia and birth weight, the former accounted for 8.7% of the variance and the latter accounted for only 4.7%. The moderate hyperbilirubinemia neonates' scores also showed a negative correlation with the autonomic system and more frequent presence of tremors. After 24 hours, a decrease in serum bilirubin within the moderate hyperbilirubinemic group was associated with improved scores. At 3 weeks of age, the behavioral assessment of the 2 groups did not show significant differences. CONCLUSIONS: Untreated moderate hyperbilirubinemia is associated with a transient and apparently reversible alteration of neonatal behavior, particularly in the social-interactive area.