Original article


Epidemiology and biomolecular characterization of carbapenem-resistant klebsiella pneumoniae in an Italian hospital

M.L. Cristina1,2, M. sartini1,2, g. Ottria1,2, E. sChinCa1,2, n. CEndErELLO2, M.P. CrisaLLi2, P. Fabbri2,

g. LO PintO2, d. UsigLiO2, a.M. sPagnOLO1

1department of health sciences, University of genoa, italy; 2galliera hospital, genoa, italy


Keywords

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Carbapenem-resistant Klebsiella pneumoniae • Bloodstream infection • Mortality


Summary

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Objective. To describe the occurrence of CRKP infections in a tertiary care hospital and to analyse the allelic profiles of the clinical strains involved and the most frequent carbapenemases. Design. The study analyzed cases of infection due to CRKP in the period 2013-2014; 147 cases were recorded, most of which (82.31%) were in-hospital infections.

Setting. A hospital in northern Italy.

Methods. We retrospectively collected: data on patient charac- teristics and the microbiological characteristics of CRKP. Iso- lates from 72 of the in-hospital cases underwent molecular typing (MLST); in addition, in each isolate, a procedure for the detection of the blaKPC gene was carried out.

Results. The in-hospital death rate was 24.0% in 2013 and 37.5% in 2014. However, the difference between these two values did not prove statistically significant (P > .05).

Analysis of mortality revealed that bloodstream infections were more frequently associated with death than other infections (c2 = 14.57, P < .001). The age-adjusted Cox proportional haz- ard model revealed that the patients with bacteremia due to CRKP had a 3-fold higher risk of death (HR 3.11; 95% CI 1.66 - 5.84, P< .001) than those with infections of other sites.

MLST revealed that the prevalent allelic profile was ST 512 (79.62%); the most frequent carbapenemase was KPC-3 (83.8%). Conclusions. Our results are in line with those of recent studies, which have shown that the spread of CRKP in Italy is a matter of concern and that further efforts have to be made to prevent the potential dissemination of carbapenemase-producing clones of K. pneumoniae, whenever possible.


Introduction


Since the 1970s, the selective pressure exerted by antibiot- ics has given rise to bacterial species that are increasingly resistant, and the last 20 years have seen a dramatic rise in the number of multi-resistant pathogenic strains [1]. Multidrug-resistant Klebsiella pneumoniae is one of the leading causes of nosocomial infection worldwide. It causes urinary tract infections (UTIs), pneumonia and in- tra-abdominal infections in hospitalized immunocompro- mised patients with severe underlying diseases [2] and is responsible for roughly 15% of Gram-negative infections in hospital intensive care units (ICUs) [3].

After the spread of strains resistant to beta-lactams at the end of the 20th century, the diffusion of isolates of K. pneumoniae resistant to carbapenems and colistin is now reducing treatment options and the containment of infec- tions [4]. In recent years, carbapenem-resistant Klebsi- ella pneumoniae (CRKP) has become a widespread con- cern and carbapenemase production mediated by blaKPC is the most prevalent mechanism conferring resistance to carbapenems [5]. Outbreaks of CRKP have increasingly been reported in various healthcare settings [4, 6, 7], in- cluding long-term acute care hospitals [8].

Risk factors for colonization and infection with CRKP are similar to those associated with other multidrug-resistant organisms [9]. Lengthy hospitalization, antibiotic use, in- vasive procedures and admission to the ICU [8] are as- sociated with an increased risk of acquisition of CRKP. Mortality rates due to infections caused by CRKP are high, ranging from 26% to 44% and reaching 70% in cases of bacteremia [10-12]. However, deaths reported to be associated with carbapenem-resistant K. pneumoniae have included several cases in which the patient had a severe underlying disease, and it is frequently difficult to determine whether carbapenem-resistant K. pneumoniae infection was the cause of death.

The aim of the present study was to describe the occur- rence of CRKP infections in a northern Italian hospital and to analyse the allelic profiles of the clinical strains involved and the most frequent carbapenemases.


Materials and methods


Setting

The study was conducted in a nationally renowned, highly specialized Northern Italian hospital organized

in accordance with treatment intensity. Structured in pa- vilions, the hospital has 458 beds (mainly located in 3- and 4-bed rooms) and each year carries out over 15,000 ordinary hospitalizations and more than 8600 medical procedures in Day Hospital and Day Surgery settings.


Study design

The study retrospectively analyzed cases of infection due to CRKP in the period 2013-2014.

Patients who were identified as having CRKP infections within the first 72 h of admission were defined as com- munity-associated cases or, if they had been exposed to healthcare settings during the previous three months, im- ported healthcare-associated cases. Clinical episodes of infection were considered to be hospital-acquired if they were not present at the time of hospital admission and appeared 72 hours after admission.

A record was made of each case patient’s age, gender, history of hospitalizations, antibiotic treatments, dura- tion of hospitalization, the date of the first CRKP de- tection, the site of infection and co-infections, invasive procedures and outcomes.

If a positive patient had been transferred from one ward to another, acquisition of the infection was attributed to the ward in which the diagnosis of infection was made, as the exact site of acquisition could not be determined. The incidence of infections was calculated per 1000 days of hospitalization.


Classification of patients according

to infection risk and type of isolation applied

Patients were grouped into two categories of infection risk: high-risk and medium-risk according to the charac- teristics of the patient and the site of infection. Patients were deemed to be at high risk if they presented one or more of the following characteristics: presence of excre- tions/secretions at the infection site, confinement to bed, lack of self-sufficiency, and great need for assistance. Patients in this category underwent structural or cohort isolation; if this was not possible, functional isolation was implemented.

Patients were defined as being at medium risk if they presented one or more of the following characteristics: presence of reduced secretions or excretions at the infec- tion site, capability of temporal and spatial orientation, ability to cooperate, self-sufficiency, and low-medium need for assistance. These patients underwent functional isolation.


Microbiologic methods

Bacterial identification and antimicrobial susceptibility testing were carried out by means of the Phoenix 100 Automated Microbiology System (Becton Dickinson Diagnostic Systems, USA).

Confirmatory MIC testing for imipenem, meropenem and ertapenem was carried out by means of Etest (bio- Mérieux SA, France) and the Kirby Bauer disk diffusion method [13]. All collected isolates that were confirmed to be non-susceptible to imipenem and/or meropenem and/or ertapenem according to the EUCAST break-

points [14] were considered to be Carbapenem-resist- ant K. pneumoniae and underwent a modified Hodge test [15] to confirm carbapenemase production. In addi- tion, in order to identify which carbapemenase was pres- ent, PCR for the blaKPC gene was carried out [16].


Biomolecular analysis

The isolates from 72 of the in-hospital cases underwent molecular typing by means of the MLST technique (http://www.pasteur.fr/recherche/genopole/PF8/mlst/ Kpneumoniae.html). This technique involves amplify- ing and sequencing seven housekeeping genes (rpoB, gapA, mdh, pgi, phoE, infB, tonB) and, through com- parison with the data available in an online databank, enables a specific ST (Sequence Type) to be assigned to each isolate. The amplification protocol prescribes an initial denaturing phase at 94°C for 5 minutes, followed by 35 cycles at 94°C for 30 seconds, 50°C for 30 sec- onds and 72°C for 30 seconds, with a final extension at 72°C for 5 minutes.

In addition, in each isolate, a procedure for the detection of the blaKPC gene was carried out through the amplifica- tion and sequencing of a DNA fragment of about 1000bp by means of the following primer pairs: blaKPC-Forward 5’-TGTCACTGTATCGCCGTC-3’ and blaKPC-Reverse

5’-CTCAGTGCTCTACAGAAAACC-3’. The amplifi-

cation phase consisted of an initial denaturing phase at 95°C for 5 minutes, followed by 35 cycles at 95°C for 60 seconds, 55°C for 40 seconds and 72°C for 90 seconds, with a final extension at 72°C for 10 minutes. The se- quences obtained were compared with those available in the NCBI database (http://blast.ncbi.nlm.nih.gov/Blast. cgi), which enabled the blaKPC gene to be characterized. For both the identification of the Sequence Type and de- tection of the blaKPC gene, sequencing was carried out by means of an Applied Biosystems 3730xl DNA Analyzer (Life Technologies, USA).


Infection control measures

In all high-risk patients, rectal swabs were taken by the ward nurse on admission to the ward. Cultures were then sent directly to the bacteriology laboratory for prompt CRKP identification.

The diffusion of the microorganism was monitored by means of continuous integrated microbiological sur- veillance, starting with laboratory data (alert organism surveillance). Following laboratory identification of an epidemiologically important microorganism, the dedi- cated software of the surveillance system automatically e-mails the data to all the members of the Hospital Infec- tions Committee, who then implement the interventions deemed necessary, with particular regard to the applica- tion of isolation measures.

A validated report is simultaneously sent through the laboratory information system to the hospital facility in- volved.

Antibiotic therapy was instituted after consultation with the infectious-disease specialist on the basis of case his- tory, patients’ clinical features, microbiological isolates and antibiotic sensitivities.

Hospital-wide policies to prevent nosocomial carbap- enem-resistant Enterobacteriaceae transmissions were introduced on the basis of an institutional protocol that was developed in accordance with the American Cent- ers for Disease Control and Prevention (CDC) indica- tions [17].


Statistical analysis

Statistical analysis was carried out by means of STATA SE13TM software (Stata Corp LP, USA). The results were analyzed in terms of descriptive statistics, and dif- ferences between groups were evaluated by means of non-parametric chi-square test and Fisher’s mid-P exact test.

Kaplan-Meier survival curves were assessed by means of a log-rank analysis, to compare overall survival, and a COX proportional hazard model, to assess the role of possible confounders. A p-value < 5% was considered significant.


Results


In the period of observation, 147 cases of CRKP infec- tion were recorded (75 in 2013 and 72 in 2014) most of which (82.31%) were in-hospital infections.

The overall mean age of the patients was 78.95 ± 12.05 years (range 26-97 years). Women accounted for 50.34% of the patients; their mean age was 81.49 ± 10.51 years (range 42-97 years). The mean age of the male patients


Tab. I. Carbapenem-resistant Klebsiella pneumoniae case characteristics.

Value

(%)

Total no. confirmed

147

Unit in which the diagnosis of infection was made

medical ward

55

(37.41)

geriatric unit

53

(36.06)

Surgical ward

30

(20.41)

ICU*

9

(6.12)

Cases from:

hospital

121

(82.31)

Other healthcare facility

13

(8.84)

Community

13

(8.84)

Invasive procedures correlated with infection

56

(46.28)

Bladder catheter

42

(75.00)

CvC§

5

(8.92)

erCp^

3

(5.36)

Tracheal intubation

2

(3.57)

percutaneous surgical drainage

2

(3.57)

Urethral catheter

1

(1.79)

pIvC°

1

(1.79)

Sites of CrKp infection

Urinary tract infection

96

(65.31)

Bloodstream infection

23

(15.64)

Surgical site infection

15

(10.20)

Airways

7

(4.76)

Other

6

(4.09)

Specimen type

Urine from catheter

65

(44.22)

Urine

31

(21.09)

Blood

25

(17.00)

Other

6

(4.08)

Bronchial aspirate

4

(2.72)

Surgical fragment

4

(2.72)

Abdominal fluid

4

(2.72)

pus

4

(2.72)

Skin swab

1

(0.68)

Wound swab

1

(0.68)

Bile

1

(0.68)

expectorate

1

(0.68)


*ICU = intensive Care Unit - §CvC = central venous catheter - ^erCp = endoscopic retrograde Cholangiopancreatography - °pIvC = peripheral Intravenous Catheter

was 76.37 ± 12.99 years (range 26-96 years). The dif- ference in age between males and females proved to be statistically significant (t = 2.6265, P < .01).

The mean duration of hospital stay was 35.32 ± 25.04 days (range 1-168).

In 46.28% of the cases of in-hospital infection, the infec- tion was related to an invasive procedure (Tab. I).

The Age-adjusted Charlson Comorbidity Index revealed comorbidity values in the hospitalized patients of 6.21 and 6.67 in 2013 and 2014, respectively (P >.05).

Over the two-year observation period, 47.62% of the CRKP-infected patients were deemed at high risk. On considering the two years separately, a statistically sig- nificant difference emerged (Χ2 = 16.0481, P < .001) between the percentage of patients classified as being at high infective risk in 2013 (32.00%) and in 2014 (63.89%).

A total of 70.77% of patients were hospitalized in 4-bed rooms, 17.69% in 2-bed rooms and 7.69% in single rooms. It was possible to hospitalize only 3.85% of patients in rooms with dedicated bathrooms (1.54% in 4-bed rooms, 1.54% in 2-bed rooms and 0.77% in single rooms).

Table I reports some characteristics of the cases of infec- tion examined: the unit in which the diagnosis of infec- tion was made, the provenance of the cases, the inva- sive procedures performed, the sites of infection and the specimen type.


Antimicrobial

No. resistant/No. tested

(%)

Aztreonam

145/147

(99)

Amikacin

45/55

(82)

Amoxicillin- clavulanic acid

145/147

(99)

Colistin

38/145

(26)

Cefalexine

91/92

(99)

Cefepime

102/147

(99)

Cefixime

91/92

(99)

Cefotaxime

145/147

(99)

Ceftazidime

145/147

(99)

Cefuroxime

144/145

(99)

Ciprofloxacin

145/147

(99)

Fosfomycin

17/130

(13)

gentamicin

25/147

(17)

Levofloxacin

55/55

(100)

moxifloxacin

91/92

(99)

Ampicillin

147/147

(100)

piperacillin

55/55

(100)

piperacillin- tazobactam

145/147

(99)

Tygecicline

5/41

(12)

Tobramycin

135/147

(92)

Thrimetoprim

76/94

(81)

Thrimetoprim- sulfamethoxazole

112/147

(76)

Antimicrobial

No. resistant/No. tested

(%)

Aztreonam

145/147

(99)

Amikacin

45/55

(82)

Amoxicillin- clavulanic acid

145/147

(99)

Colistin

38/145

(26)

Cefalexine

91/92

(99)

Cefepime

102/147

(99)

Cefixime

91/92

(99)

Cefotaxime

145/147

(99)

Ceftazidime

145/147

(99)

Cefuroxime

144/145

(99)

Ciprofloxacin

145/147

(99)

Fosfomycin

17/130

(13)

gentamicin

25/147

(17)

Levofloxacin

55/55

(100)

moxifloxacin

91/92

(99)

Ampicillin

147/147

(100)

piperacillin

55/55

(100)

piperacillin- tazobactam

145/147

(99)

Tygecicline

5/41

(12)

Tobramycin

135/147

(92)

Thrimetoprim

76/94

(81)

Thrimetoprim- sulfamethoxazole

112/147

(76)

Tab. II. Antibiogram for carbapenem-resistant Klebsiella pneumo- niae cases reported.

The incidence of infections was 0.442 pts/1000 days of hospitalization in 2013, and 0.513 pts/1000 days of hos- pitalization in 2014 (P > .05).

In the entire period of observation, we recorded 24 cases of coinfection due to: Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus au- reus, Acinetobacter baumannii, Enterococcus faecium, Proteus mirabilis, Corynebacterium striatum, Entero- coccus casselliflavus, Moraxella morganii, Serratia marcescens, and Staphylococcus haemolyticus. Among these microorganisms, we identified ESBL-positive strains of E. faecium, E coli, P. mirabilis and P. aeru- ginosa; VRE strains of E. casseliflavus and E. faecalis, and strains of MRSA.

With regard to patient outcomes over the two years of observation, 30.61% of the patients died in hospital; 12.24% were transferred to other units in the hospi- tal; 0.68% were transferred to other hospitals; 19.73% were transferred to a residential facility; 4.76% were discharged with home assistance, and 31.97% were dis- charged home without assistance.

The in-hospital death rate was 24.0% in 2013 and 37.5% in 2014. However, the difference between these two val- ues did not prove statistically significant (P > .05).

Analysis of mortality by means of Kaplan-Meier sur- vival curves revealed that bloodstream infections were more frequently associated with death than were urinary infections and other infections (Fig. 1), the difference being statistically significant (Χ2 = 14.57, P < .001).

Of the 23 cases of bloodsteam infections, 14 (12 hospital and 2 community) were cases of primary bacteremia; 10 of these patients died. The remaining 9 cases were sec- ondary bacteremia, all nosocomial; 5 of these patients died. The mean age of the patients with primary bactere- mia was 75.36 ± 10.70 years (range 58-90), while that of the patients with secondary bacteremia was 77.89 ± 9.57 years (range 63-92).

image

The age-adjusted Cox proportional hazard model re- vealed that the patients with bacteremia due to CRKP


Fig. 1. Kaplan-meier curves of survival probability of patients with carbapenem-resistant Klebsiella pneumoniae infection, by infec- tion site.

Fig. 1. Kaplan-meier curves of survival probability of patients with carbapenem-resistant Klebsiella pneumoniae infection, by infec- tion site.


had a 3-fold higher risk of death (Hazard ratio [HR], 3.11; 95% CI 1.66-5.84, P < .001) than those with infec- tions of other sites.

With regard to the results of antimicrobial susceptibil- ity testing, the data on the resistance of the strains are reported in Table II; 26% of strains proved resistant to colistin. The antimicrobial ertapenem was the most fre- quently reported carbapenem tested for susceptibility (100%), followed by meropenem (98.6%) and imipenem (48.3%).

Genotyping of the strains by means of MLST revealed that the prevalent allelic profile was ST 512 (79.62%), followed by ST 307 (8.97%), ST 101 (3.85%), ST 147

(3.85%), ST 258 (3.85%), ST 15 (1.28%), and ST 45

(1.28%). Detection of the blaKPC gene revealed that the most frequent carbapenemase was KPC-3 (83.8%) and that KPC-2 was less common (16.2%).

With regard to blaKPC gene detection in relation to the al- lelic profiles, it emerged that STs 258, 101, 147, 15 and 45 displayed only blaKPC-2; that ST 307 were associated only to blaKPC-3; that most ST 512 hosted the blaKPC-3 gene (98%), and that only a small portion hosted blaK- PC-2 (2%) (Fig. 2).


Discussion and conclusions


The emergence and spread of Klebsiella pneumoniae harboring carbapenemases have given rise to several problems regarding infection control and treatment.

Carbapenamase-associated resistance is alarming for a number of reasons. The presence of these enzymes, in addition to signifying resistance to carbapenems, is also associated with additional mechanisms of resis- tance to other antibiotic classes which, together, result in microbes that are highly multidrug resistant and in some cases panresistant [18-20]. Consequently, they are invariably associated with high treatment failure rates [21].

CRKP has rapidly become a major health concern for hospitalized patients in industrialized countries, and in- fection rates have been dramatically increasing world- wide over the past 10 years. The first case of carbapen- emase-producing Klebsiella pneumoniae in Italy was

detected in October 2008. In 2011, the European Anti- microbial Resistance Surveillance Network (EARS-Net) reported that Italy was one of the most seriously affected countries in Europe, with a worrisome increasing trend in CPKP [22-24].

image

Fig. 2. Percentage distribution of carbapenemases in the various allelic profiles isolated

Fig. 2. Percentage distribution of carbapenemases in the various allelic profiles isolated

These strains are implicated in nosocomial outbreaks and cause serious infections in ICUs. Moreover, recent national data have shown that CRKP is more frequently isolated from patients outside ICUs, and often from those admitted to geriatric or internal medicine wards [25, 26]. In the present study, we documented the occurrence of 147 cases of CRKP infections in a Northern Italian hos- pital. These cases were chiefly detected in medical wards (37.41%) and geriatric units (36.06%), followed by sur- gical wards (20.41%) and ICUs (6.12%). These wards are also frequently involved in CRKP infections in other countries. Indeed, Poulou et al. [27] reported that, of the 73 CRKP infections registered between 2009 and 2011 at a university hospital in Greece, 43.8% were identified in the ICU, 41.1% in medical wards and 15.1% in sur- gical wards. Moreover, Kanerva described an outbreak of carbapenemase-producing Klebsiella pneumoniae in a primary care hospital in Finland; this was confined to one geriatric ward and involved 142 patients with a mean age of 83 years [28].

Most of the CRKP-positive cases described in the present study involved elderly patients (mean age 78.95 ± 12.05 years). The Age-adjusted Charlson Comorbidity Index revealed values of underlying comorbidity in hospital- ized patients of 6.21 in 2013 and 6.27 in 2014 (P > .05), reflecting a high level of complexity of assistance, which remained fairly constant throughout the observation pe- riod.

These findings are in line with those of recent studies, which have shown that the spread of CRKP in Italy is becoming a matter of concern in areas of care that were generally considered to be at lower risk. Moreover, one of the principal targets of CRKP is the population of ge- riatric patients [24, 29, 30], who display a high degree of clinical complexity and a large number of comorbidities and are frequently bedridden or cognitively impaired. Thus, frailty and comorbidity are, in themselves, a major risk factor for CRKP colonization, together with those already described in the literature (length of hospitaliza- tion, number of previous hospitalizations and/or previ- ous ICU stays, previous antibiotic use, severity of ill- ness, etc) [21, 30, 31].

In a large, retrospective, matched (1:2) case-control study in five Italian hospitals, Tumbarello et al. identified risk factors for CRKP infections; the strongest predictor of CRKP isolation was a history of ≥ 2 previous acute-care hospitalizations in the year before the index culture. Iso- lation was also associated with indwelling medical de- vices, such as urinary catheters, central venous catheters (CVCs) and surgical drains [21]. Invasive procedures are well-known risk factors for infection by CRKP [32]; indeed, the formation of biofilms on these devices is im- portant in the pathogenesis of these bacteria [33, 34]. In 46.28% of the cases of in-hospital infection recorded in the present study, it was possible to correlate the infec-

tive event with an invasive procedure. Specifically, 75% of these cases occurred in patients with a urinary cath- eter; and indeed, the principal specimen type from which CRKP was isolated was catheter urine (44.22%). Lower percentages of infections related to invasive procedures were associated with CVCs (8.92%), endoscopic retro- grade cholangiopancreatography (5.36%), and various other indwelling medical devices (overall, 10.72%).

During the two-year observation period, we registered an increase in the incidence of new clinical cases per 1,000 patient days. Fortunately, however, this increase did not reach statistical significance (0.442 cases/1,000 patient days in 2013 versus 0.513 cases/1,000 patient days in 2014; P>.05), despite the fact that a statistically significant difference (c2 = 16.0481, P < .001) emerged between the percentages of patients classified as be- ing at high infective risk in 2013 (32.00%) and in 2014 (63.89%).

Following laboratory identification of CRKP, the mem- bers of the Hospital Infections Committee implemented the interventions deemed necessary, with particular re- gard to the application of isolation measures, the disin- fection of environmental surfaces and improvement of hand hygiene compliance. Indeed, eliminating surface contamination as a source of patient-to-patient trans- mission of nosocomial pathogens requires multiple in- terventions aimed at cleaning/disinfecting the environ- ment and improving adherence to hand hygiene guide- lines [35-37].

The mortality rate due to all causes of infection was 24% in 2013 and 37.5% in 2014. This increase, albeit not sta- tistically significant, may have been due to the greater complexity of patients in the second year of observa- tion. In any case, the mortality rates, however evaluated, proved to be in line with the literature data, which report mortality rates between 26% and 44 % [12, 38, 39].

Moreover, it emerged that the infective event most fre- quently associated with death was bacteremia; this is in agreement with the results of previous studies; in a matched retrospective, historical cohort design study in- volving 319 patients with infections due to carbapenem- resistant K. pneumoniae, Borer et al. [40] found a similar mortality risk ratio: 3.3 (95% CI 2.9-28.5) among case subjects with carbapenem-resistant K. pneumoniae bac- teremia.

The results of our study revealed that 26% of strains proved resistant to colistin, a lower value than those re- ported in similar studies [41, 42].

Regarding the results of genotyping, it emerged that the allelic profile most frequently observed in the hospital was ST 512; this is in line with the results of other stud- ies conducted in Italy [43]. ST 512 (allelic profile: 54- 3-1-1-1-1-79) is a single-locus variant of ST 258 (allelic profile: 3-3-1-1-1-1-79) and is the clone most frequently associated worldwide with the spread of KPCs.

The ST 512 detected in the present study mainly pro- duces KPC-3 carbapenemase; the blaKPC-3-containing strain of K. pneumoniae displays an exceptional combi- nation of multidrug resistance, virulence and ability to spread [44] and the KPC-3-producing K. pneumoniae

ST 512 clone has emerged as a successful new lineage, capable of disseminating KPC-3 in Europe [45].

The results of the present study revealed that the charac- teristics of the predominant strain, together with the high levels of comorbidity of the patients involved and the difficulty of ensuring structural isolation owing to the small number of dedicated rooms, have, at least in part, undermined the success of the measures for prevention and control adopted in the hospital.


Acknowledgments


The study was supported by a grant from the University of Genoa (University Research Project Grant). The au- thors declare no conflict of interest.


Authors’ contributions


MLC conceived, designed and coordinated the research. NC, PF and DU collected data.

DU and GO performed respectively microbiological and biomolecular analyses. ES performed the data quality control.

MS optimized the informatics database and performed the statistical analyses. MPC and GLP evaluated the re- sults. MLC and AMS wrote the manuscript.

All Authors revised the manuscript and gave their con- tribution to improve the paper. All authors read and ap- proved the final manuscript.


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n Received on June 9, 2016. Accepted on August 29, 2016.


n Correspondence: Marina Sartini, Department of Health Sciences, University of Genoa, via Pastore, 1, 16132 Genova, Italy - Tel. +39 010 3538447 - Fax +39 010 3538216 - E-mail: sartini@unige.it