Next Article in Journal
Fabrication and Evaluation of W/O Emulsion Loaded with Linum usitatissimum Seeds Extract for Anti-Leishmaniasis Efficacy
Next Article in Special Issue
Genetic Characterization of Carbapenem-Resistant Klebsiella spp. from Municipal and Slaughterhouse Wastewater
Previous Article in Journal
Antimicrobial Activity and 70S Ribosome Binding of Apidaecin-Derived Api805 with Increased Bacterial Uptake Rate
Previous Article in Special Issue
Comparison of Six Phenotypic Assays with Reference Methods for Assessing Colistin Resistance in Clinical Isolates of Carbapenemase-Producing Enterobacterales: Challenges and Opportunities
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Antibiotics
Volume 11
Issue 4
10.3390/antibiotics11040431
antibiotics-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Prevalence and Molecular Typing of Carbapenemase-Producing Enterobacterales among Newborn Patients in Italy

by
Marilena Agosta
1,†,
Daniela Bencardino
2,†,
Marta Argentieri
1,
Laura Pansani
1,
Annamaria Sisto
1,
Marta Luisa Ciofi Degli Atti
3,
Carmen D’Amore
3,
Lorenza Putignani
4,
Pietro Bagolan
5,
Barbara Daniela Iacobelli
5,
Andrea Dotta
6,
Ludovica Martini
6,
Luca Di Chiara
7,
Mauro Magnani
2,
Carlo Federico Perno
1,
Francesca Andreoni
2,‡ and
Paola Bernaschi
1,*,‡
1
Microbiology and Diagnostic Immunology Unit, Department of Diagnostic and Laboratory Medicine, Bambino Gesù Children’s Hospital, IRCCS, 00165 Rome, Italy
2
Department of Biomolecular Sciences, University of Urbino “Carlo Bo”, 61032 Fano, Italy
3
Clinical Pathways and Epidemiology Unit, Bambino Gesù Children’s Hospital, IRCCS, 00165 Rome, Italy
4
Human Microbiome Unit, Department of Diagnostics and Laboratory Medicine, Bambino Gesù Children’s Hospital, IRCCS, 00165 Rome, Italy
5
Neonatal Surgery Unit, Medical and Surgical Department of the Fetus-Newborn-Infant, Bambino Gesù Children’s Hospital, IRCCS, 00165 Rome, Italy
6
Neonatal Intensive Care Unit, Medical and Surgical Department of the Fetus-Newborn-Infant, Bambino Gesù Children’s Hospital, IRCCS, 00165 Rome, Italy
7
Pediatric Cardiac Intensive Care Unit, Department of Cardiology and Cardiac Surgery, Bambino Gesù Children’s Hospital, IRCCS, 00165 Rome, Italy
*
Author to whom correspondence should be addressed.
†
Co-first author: These authors contributed equally to this work. Author order was determined alphabetically.
‡
Co-last author: These authors contributed equally to this work. Author order was determined alphabetically.
Antibiotics 2022, 11(4), 431; https://doi.org/10.3390/antibiotics11040431
Submission received: 1 March 2022 / Revised: 18 March 2022 / Accepted: 21 March 2022 / Published: 23 March 2022
(This article belongs to the Special Issue Carbapenemase-Producing Enterobacterales)
Browse Figures
Versions Notes

Abstract

:
The spread of carbapenemase-producing Enterobacterales (CPE), especially Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli), is a serious public health threat in pediatric hospitals. The associated risk in newborns is due to their underdeveloped immune system and limited treatment options. The aim was to estimate the prevalence and circulation of CPE among the neonatal intensive units of a major pediatric hospital in Italy and to investigate their molecular features. A total of 124 CPE were isolated from rectal swabs of 99 newborn patients at Bambino Gesù Children’s Hospital between July 2016 and December 2019. All strains were characterized by antimicrobial susceptibility testing, detection of resistance genes, and PCR-based replicon typing (PBRT). One strain for each PBRT profile of K. pneumoniae or E. coli was characterized by multilocus-sequence typing (MLST). Interestingly, the majority of strains were multidrug-resistant and carried the blaNDM gene. A large part was characterized by a multireplicon status, and FII, A/C, FIA (15%) was the predominant. Despite the limited size of collection, MLST analysis revealed a high number of Sequence Types (STs): 14 STs among 28 K. pneumoniae and 8 STs among 11 E. coli, with the prevalence of the well-known clones ST307 and ST131, respectively. This issue indicated that some strains shared the same circulating clone. We identified a novel, so far never described, ST named ST10555, found in one E. coli strain. Our investigation showed a high heterogeneity of CPE circulating among neonatal units, confirming the need to monitor their dissemination in the hospital also through molecular methods.

1. Introduction

The spread of carbapenem-resistant Enterobacterales (CRE) is an emerging concern worldwide and, noteworthy, carbapenem-resistant gram-negative bacterial infections are counted in the WHO priority pathogen list [1,2]. Due to continuous evolution of molecular mechanisms of resistance, the epidemiology of CRE is changing, and an increasing number of people are being colonized and infected by these organisms worldwide [3,4]. These infections are associated with high mortality ranging from 40% to 50%, and their prevalence continue to increase [4]. Indeed, carbapenemase-producing Enterobacterales (CPE) typically carry genes that confer resistance to other antibiotics and they are often extensively drug-resistant or even pan-drug resistant, limiting treatment options and leading to a high rate of therapeutic failures and subsequently to fatalities [5,6]. Globally, Klebsiella pneumoniae carbapenemases (KPCs) are the most common transmissible genes circulating in Enterobacterales. They have the ability to hydrolyze all β-lactams, and strains carrying blaKPC often acquire resistance to several antibiotic agents, resulting in Multidrug-Resistant Organisms (MDROs) [7]. The rapid spread of KPC-Enterobacterales is due to the successful ST258 lineage, multidrug-resistant strains of K. pneumoniae endemic in several countries and responsible for many outbreaks [8].
However, in the last decade, the emergence of the most recently described carbapenemase New Delhi metallo-lactamase (NDM-1) was widely described as a matter of concern in clinical settings. This carbapenemase belongs to the metallo-lactamases class (MBLs) hydrolyzing a broad range of β-lactams with the exception of monobactams such as aztreonam. Infections caused by NDM producers include urinary tract infections, peritonitis, septicemia, pulmonary infections, soft tissue infections, and device-associated infections [9]. In contrast with KPC genes, the spread of NDM-type MBLs seems to be not associated with clonal lineages, but it is mediated by different plasmid incompatibility (Inc) groups [4,9]. Indeed, the clonal spread of these microorganisms among different patients develops very easily, and resistance genes to carbapenems can be transmitted between microorganisms of different species through plasmids [10].
In this scenario, the spread of (MDROs) is particularly worrisome in the Neonatal Intensive Care Unit (NICU): in this complex care setting, the emergence and dissemination of CRE and CPE, especially K. pneumoniae and E. coli, can occur with particular frequency and serious risks for newborns [11,12,13]. The epidemiological context and, in particular, the level of endemicity in communities and care settings, play a very important role in the risk of importing isolates and/or genetic resistance determinants into a NICU. Community-acquired (CA) pathogens are introduced in the hospital through the patients (including neonates and infants) themselves, their parents, and healthcare professionals. Furthermore, in the reference NICU of a specific territorial area, the epidemiology of microorganisms is strongly influenced by patient movements from and to other NICUs. Alongside the condition for which they are transferred (e.g., malformations, surgical emergencies, etc.), they present additional risk factors for the carriage of MDRO, i.e., previous exposure to another high-risk care setting [14,15,16,17].
On the other hand, prolonged hospitalization in such fragile patients is known to predispose to hospital-acquired colonization (HAC) [18,19]. For these reasons, to monitor the spread of carbapenemase-producing organisms (CPO), different international institutions have developed numerous guidelines [20,21,22], which have also been adopted in Italy.
Current prevention strategies for carriage and infections by CPE include active surveillance using culture and/or molecular methods, adoption of contact precautions, isolation or cohorting and, in selected cases, decolonization. Several studies emphasize the importance of identifying individuals carrying antimicrobial-resistant bacteria in both the patient and healthy population [23,24]. In fact, asymptomatic rectal carriers are considered the main source of spread of multidrug-resistant organisms, especially among fragile individuals, such as pediatric ones, where colonization is a precursor to infections such as bacteraemia, pneumonia, central nervous system (CNS), and urinary tract infections [25,26]. A large amount of information is reported in the literature for adults but, to date, little is known about the epidemiology and screening strategy of CPE in children, and even less in newborns.
The aim of this study was to estimate the distribution of CPE strains isolated during a three-year period from newborn patients admitted to the NICU and cardiac intensive care unit (CICU) of a large, tertiary care pediatric Italian hospital. All CPE strains were characterized in terms of their antimicrobial resistance patterns and molecular features such as determinants and plasmid replicons. Each strain of K. pneumoniae and E. coli, representative for each PBRT profile, was further characterized by multilocus-sequence typing (MLST).

2. Results

2.1. Epidemiological Features

Between July 2016 and December 2019, 124 CPE strains were consecutively collected from 99 newborn patients (44 females and 55 males) aged from six days to one year. A large part of newborns (72%) was hospitalized in the neonatal surgical unit (NSU), 15% in neonatal intensive therapy (NIT), 6% in the sub-intensive neonatal unit (SNU), and the remaining 5% in the cardiac intensive care unit (CICU). All strains were collected from rectal swabs: 105 (84%) were isolated from Italian patients, whereas 19 (15.3%) from 14 foreign patients: three from Burundi, two from Libya, Russia, and Romania, and one from Iraq, Georgia, Central African Republic, Sierra Leone, and Ukraine. From all patients, at least one strain was isolated (one strain from each swab), with the exception of 25 children subjected to a second swab during the hospitalization stay or after the second admission. In detail, 50 strains were isolated from 25 patients, and the remaining 74 strains were isolated from 74 patients. Moreover, the major part of the strains (n = 103; 82%) were isolated from patients colonized during their hospitalization, whereas 21 strains (17%) were community-acquired because they were isolated from patients who were colonized at the time of their admission to the hospital.

2.2. Isolate Identification

Of 124 CPE strains, K. pneumoniae (n = 55; 44%), E. coli (n = 44; 35%), Klebsiella oxytoca (n = 7; 6%), Enterobacter cloacae (n = 7; 6%), Citrobacter freundii (n = 6; 5%), Klebsiella aerogenes (n = 2; 2%), Citrobacter koseri (n = 1; 1%), Serratia marcescens (n = 1; 1%), and Morganella morganii (n = 1; 1%) were identified. The most prevalent species of K. pneumoniae and E. coli were widely detected both in NICU and CICU wards, with a significant presence in NSU (n = 41/55 for K. pneumoniae; n = 35/44 for E. coli). One foreign patient carried Citrobacter koseri and another carried Morganella morganii.

2.3. Antimicrobial Resistance Characterization

All CPE strains were found to be resistant to nearly all antibiotics tested in this study, including imipenem (n = 121; 97%) and meropenem (n = 119; 96%). High resistance was detected towards amoxicillin–clavulanic acid (100%), cefotaxime (100%), ceftazidime (100%), and piperacillin–tazobactam (100%). The lowest rate of resistance was recorded for tigecycline (n = 4; 3%). All strains of K. pneumoniae were resistant to all the antibiotics tested, whereas those of E. coli were susceptible only to tigecycline. Among 21 strains related to community-acquired colonization, 12 different antibiotic patterns were recorded, showing very high variability.
All 124 CPE strains were positive for carbapenemase genes, with the prevalence of 73% for the New-Delhi Metallo-β-lactamase gene (blaNDM). In detail, the blaNDM gene was detected in combination with blaKPC and blaVIM in 24 and 2 strains, respectively. By contrast, the remaining blaKPC (n = 15), blaVIM (n = 13), and blaOXA-48 (n = 3) genes were individually detected.

2.4. Replicon Typing

A summary of replicons detected among the 122 CPE strains is given in Figure 1. Overall, only two strains of E. cloacae were not typeable by the PBRT, and IncA/C was the most common Inc group (65%), followed by IncFIA, IncFII, and IncFIIK found in 43%, 39%, and 32% of isolates, respectively. In this study, 18 out of 30 replicons were identified, whereas IncHI1, IncI2, IncB0, IncP1, IncW, IncI1γ, IncFIIS, IncN2, IncX1, IncX2, IncK, and IncX4 were not detected.
The majority of isolates (n = 94/122; 77%) were characterized by the multireplicon status carrying three or more different Inc groups. In detail, the predominant multireplicon profiles were FII, A/C, FIA (n = 19; 15%) and FII, A/C, FIB (n = 11; 9%), both found only among E. coli strains. Instead, the two multireplicon profiles consisting of A/C, R, FIIK, FIB KQ and FIB KQ, FIIK, R, A/C, and FIA were the most common in K. pneumoniae.
In our study, we observed that all forty-nine PBRT patterns were species-specific with few exceptions. In fact, the profile composed by the single replicon A/C was found in E. coli, E. cloacae, M. morganii, K. aerogenes, and K. oxytoca, whereas the pattern IncFIB KQ, IncFIIK was found in K. pneumoniae and C. freundii.

2.5. Correlation between PBRT and Antibiotic Resistance Patterns

The distribution of PBRT and antibiotic resistance within the collected strains is reported in Table 1. Although we detected a consistent heterogeneity, 12 (24%) PBRT profiles seemed to be related with specific antibiotic-resistance patterns and the associated carbapenemase-resistance genes. For instance, the PBRT profile IncFII, IncA/C, IncFIA (indicated with the number 5) was present in 18 E. coli strains, and all of them reported the same antibiotic resistance pattern (AMK-AMC-FEP-CTX-CAZ-CIP-GEN-IPM-TZP-MEM) and the same carbapenemase determinant (blaNDM). This type of analysis was performed for all PBRT profiles, which included at least two strains, and numbered as 2, 13, 14, 16, 17, 19, 24, 28, 33, 46, and 49 (Table 1). Moreover, within the PBRT profiles IncFII, IncA/C, IncFIA and IncFIB KQ, IncFIIK, IncR, IncA/C, and IncFIA (numbered 5 and 17, respectively) we observed the highest number of strains that not only shared the same antibiotic-resistance pattern and related determinants, but also the same species (E. coli and K. pneumoniae, respectively), origin of patients (Italy), and type of colonization. By contrast, the highest variability was found for the PBRT profile IncA/C (number 12) wherein, out of eight strains, five different species (E. coli, K. aerogenes, K. oxytoca, M. morganii, and E. cloacae), five different antibiotic-resistance patterns, and three different carbapenemase determinant patterns were recorded.
Overall, correlations between replicons and multiple resistance profiles found in this study confirmed that plasmids are an important reservoir for the spread of resistance in Enterobacterales.

2.6. Multilocus-Sequence Typing

MLST analysis performed on 39 strains, each representative of a PBRT profile found in E. coli (n = 11) or K. pneumoniae (n = 28), revealed 22 STs. As described in Table 1, 14 different STs in 28 carbapenemase-producing K. pneumoniae isolates were found, with the prevalence of ST307 (n = 5; 18%), followed by ST17 (n = 4; 14%) and ST395 (n= 4; 14%). Among four K. pneumoniae strains isolated from foreign-born neonates, four different STs were detected (ST307, ST11, ST323, and ST1412). All strains were isolated from patients colonized upon their admission (CAC), with the exception of the strain belonging to ST323 related to hospital-acquired colonization. Moreover, these patients were hospitalized in NIT and NSU wards. Despite the modest size of collection, these findings suggest a certain degree of heterogenicity, due to the high number of circulating clones independently by origin of patient and hospitalization ward.
In light of this, we constructed phylogenetic trees in order to investigate the relationship among all STs identified in our analysis, and clonal relatedness of strains is shown in Figure 2. This analysis involved 28 nucleotide sequences for K. pneumoniae with 3018 positions in the final dataset.
Only the two strains belonging to ST323 and ST1164 seemed to be less related with the others, whereas strains typed with the most common identified ST307 showed higher phylogenetic relation with strains belonging to ST35 and ST466. Thus, all K. pneumoniae strains of our collection showed a clonal relatedness with the exception of two strains that were not genetically related with the main cluster. All strains grouped in this main cluster are well-known to be responsible for carbapenem resistance spread.
On the other hand, eight different STs were observed among 11 carbapenemase-producing E. coli, and ST131 was the main, found in three strains (two isolated from foreign patients). Instead, two strains belonged to ST69, and the remaining STs (ST617, ST80, ST74, ST162, ST167, and the novel ST10555) were individually found. Moreover, all foreign patients were hospitalized in the NSU ward, and four out of five strains isolated from them belonged to different STs (ST131, ST617, ST167, and ST69), reporting community-acquired colonization. By contrast, all E. coli strains isolated from Italian patients were associated with hospital-acquired colonization concerning not only the NSU ward but also the other wards.
Notwithstanding the low number of strains typed by MLST, we detected a wide diversity of STs in our E. coli collection as confirmed by the phylogenetic analysis. It involved 11 nucleotide sequences with a total of 3423 positions in the final dataset and revealed a higher relation among strains belonging to ST131, the novel ST10555, ST80, and ST74 (Figure 2). Of note, a main cluster was not identified, but we observed an interesting similarity of the novel ST10555 with the most common ST131. The latter is known to be an emerging carbapenemase-producing clone, suggesting the ability of the novel ST10555 to disseminate carbapenemase genes.

2.7. The Novel ST10555

Interestingly, a new sequence type was identified among E. coli clinical isolates (named E. coli_104) as shown in Table 1 and Figure 2. This strain was isolated from an Italian male patient (five months of age) in 2017. The isolate exhibited the A/C replicon and the following antibiotic-resistance profile AMK-AMC-FEP-CTX-CAZ-CIP-GEN-IPM-TZP-SXT-MEM; carbapenem resistance was confirmed by the detection of blaNDM gene. It revealed a novel allelic variant for the recA gene (recorded as 772) confirmed through whole-genome sequencing. Successively, the entire genome was submitted to EnteroBase database at https://enterobase.warwick.ac.uk/species/index/ecoli (accessed on 21 March 2022), resulting in the associated ST10555 (adk 13, fumC 52, gyrB 10, icd 14, mdh 17, purA 25, recA 772). Moreover, we used genomic analysis tools to explore the whole-genome sequencing data. Through ResFinder 4.1 we confirmed the resistance observed phenotypically and found additional resistance towards tobramycin, cefoxitin, ampicillin, and ertapenem. The same analysis revealed the following resistance genes: aac(6′)-Ib3 (amikacin, tobramycin), aph(3′)-VI (amikacin), rmtC (amikacin, gentamicin, tobramycin, kanamycin), blaCMY-6 (amoxicillin, amoxicillin–clavulanic acid, ampicillin, cefotaxime, ceftazidime, piperacillin–tazobactam), blaNDM-1 (amoxicillin, amoxicillin + clavulanic acid, ampicillin, cefepime, cefotaxime, cefoxitin, ceftazidime, ertapenem, imipenem, meropenem), and blaCTX-M-15 (amoxicillin, ampicillin, cefepime, cefotaxime, ceftazidime, piperacillin). Moreover, through PlasmidFinder 2.0 and pMLST-2.0 Server, we confirmed that this isolate belonged to IncC group and to the profile IncA/C PMLST, respectively.
Finally, SerotypeFinder 2.0 Server revealed the serotype H6 encoded by the flic gene (99.94% of identity) as well as the serotype O2 encoded by wzy and wzx genes, with the identities of 98.03% and 98.65%, respectively. The genetic features of this newly emerged strain type highlight the risk associated with its circulation, especially in terms of antibiotic resistance spread that is a matter of concern in the neonatal population.

3. Discussion

Although CPE infections are endemic in Italy and neonates and infants are considered patients at high risk, few studies have been carried out to evaluate their dissemination among neonatal intensive units [12,13,27,28,29]. Retrospective studies carried out among the NICUs of different pediatric Italian hospitals reported high rates of colonization by strains with important molecular features, highlighting the role of risk factors on infection incidence. For instance, Montagnani and colleagues confirmed the immunosuppressive state of children with hematologic/oncologic conditions, invasive devices, history of surgery or hospitalization, and prior use of antibiotics as important risk factors associated with CPE infections [27]. Instead, Mammina and colleagues showed that feeding by formula was significantly associated with colonization due to cross-contamination and poor infection control procedures [30]. On the other hand, a more recent study demonstrated that the efficacy of screening programs associated with proactive measures to control cross-contamination and hospital-acquired colonization are effective to reduce the spread of CPE also in the pediatric setting [13]. Moreover, these studies were useful also to collect information about the emerging clones circulating among neonatal wards, tracing the evolution of their molecular aspects.
Here, we described CPE distribution in neonatal wards, including NICU, and a CICU of a major tertiary pediatric referral hospital in Italy, where foreign patients are also frequently admitted. The first interesting point was the high variability of CPE detected in terms of isolates and associated molecular features. In accordance with other studies, within the variety of species detected in our collection and widely associated with neonatal colonization, K. pneumoniae and E. coli were the most common [12,13,31,32,33]. Most strains (76%) exhibited resistance to at least one agent of three or more antimicrobial categories tested in our panel. Hence, they were classified as multidrug-resistant (MDR) as described by Majorakos et al. [34], increasing the risk of treatment failure. To note, almost all strains were susceptible to tigecycline (97%). This was in agreement with results reported by the SENTRY surveillance program where a good activity of tigecycline against these microorganisms was indicated [35]. Conversely, other studies reported higher levels of resistance towards this antimicrobial agent [27], and recent investigations suggest an improved efficacy of tigecycline against carbapenem-resistant Enterobacterales in combination with colistin [36] or with ceftazidime–avibactam [37]. These findings suggest the need to monitor the use of this or other last-resort antibiotics in order to limit the impairment of treatment due to increased levels of resistance.
Moreover, CPE strains have the potential to spread widely due to the localization of carbapenemase genes on mobile genetic elements, which further complicate the treatment, increasing mortality and morbidity rates [38,39]. Overall, 26% of strains coharbored a combination of two resistance genes, thus contributing to increased risk in terms of public health. It was not surprising that the predominant determinant of carbapenem resistance found in our study was blaNDM. Indeed, the rapid dissemination of New Delhi metallo-beta-lactamase (NDM)-producing carbapenem-resistant Enterobacterales have been largely detected in central Italy in the last years [40]. This spread of NDM-producing strains confirms a significant change in CPE epidemiology consisting in replacement of previous endemicity of blaKPC genes.
It is well-known that K. pneumoniae and E. coli are the main drivers of this rapid and consistent spread, and the dissemination of NDM is generally mediated by plasmids with a variety of replicon types [41]. In accordance with the literature, the predominant IncA/C plasmid (66%) detected in the present study was strongly associated with blaNDM genes [42,43]. This plasmid type, identified in all enterobacterial species, possesses a broad host range of replication and has been identified not only from human but also from animal isolates [44,45]. For all of these reasons, the IncA/C-type plasmid is considered an increasing threat to public health. Moreover, the majority of isolates (77%) carried three or more replicons defining the multireplicon-status, with a high percentage of the IncF family that it is known to be restricted to Enterobacterales. The multireplicon status promotes acquisition of plasmids carrying incompatible replicons, enlarging the host range replication also between different species [46]. A typical multireplicon IncF plasmid carries FII, FIA, and FIB, as also observed in our collection, confirming their large diffusion among clinical Enterobacterales [46,47]. From a functional point of view, FII is silent, whereas the activity of FIA as well as that of FIB is exclusively related to enteric bacteria [46].
Additional interesting information about the diversity of strains investigated in the present study was revealed by multilocus-sequence typing analysis. The identification of ST was performed for a single strain representative for each PBRT pattern in order to obtain preliminary information about the clones circulating in the neonatal intensive unit. Of all the obtained STs, ST307 was the most common in K. pneumoniae, confirming the recent and rapid spread of this emerging clone in Italy [48,49,50]. Noteworthy, the diffusion of ST307 in Italy traces the evolution of the current epidemiological change because it seems to replace the global ST258 and its endemic Italian variant ST512. Indeed, after the first outbreak occurred in Sicily (Italy) in 2008 [51], where KPC-3-producing K. pneumoniae ST258 was mainly responsible, a second surveillance carried out in 2014 revealed the emerging of KPC-3-producing strains ST307 [29,52]. As also detected in our investigation, ST307 is a novel distinctive lineage carrying blaKPC determinants [49], even if in our study we found it in association with blaNDM or blaOXA-48, suggesting the potential to acquire advantageous features for the adaption to clinical niches. Furthermore, the increased detection of ST307 also in other countries in the last years suggests not only its pivotal role in the spread of antibiotic resistance but also the potential to become one of the most clinically relevant clones. Our data confirmed the increasing CPE prevalence in neonates and infants and showed that the molecular characteristics of strains are evolving. Moreover, CPE colonization represents an important reservoir for nosocomial infections due to their stronger virulence and transmission. Hence, an active screening also through molecular typing is very useful to classify genotypes and is a priority in vulnerable patients like newborns, where multiple-antibiotic resistance can result in failed infection treatment.
On the other hand, E. coli strains characterized by MLST revealed several STs, but this collection was too limited, and the frequency of these clones could be underestimated. Among them, ST131, known to be responsible for hospital- and community-acquired urinary tract infections as well as bloodstream infections, was most commonly detected. Most strains belonging to ST131 are MDR, therefore limiting therapeutic options that cause recurrent infections [53].
The identification of the novel ST10555 highlights the variability observed among E. coli strains despite the low size of collection. It was isolated from an Italian patient and exhibited resistance towards all of antibiotics tested in our study, with the exception for tigecycline. Whole-genome analysis revealed the carriage of other determinants besides blaNDM, confirming the risk associated with its distribution among newborn patients. This novel ST was phylogenetically related with ST131, described as a high-risk clone due to its role in antibiotic resistance spread, confirming the continuous emergence of new clones with potential to colonize clinical niches (Figure 2).
Notwithstanding the limited size of our collection, the richness and variability of genetic repertoire showed by these strains made more evident the dynamic evolution of CPE and, consequently, the importance of monitoring their distribution in healthcare settings.
From a health perspective, the results of this study highlight the urgency of addressing the surveillance of CPE distribution in pediatric hospitals. One important challenge for hospitals consists of collaborative efforts with different and external professional figures of health sciences. Indeed, in addition to the commonly reported guidelines implemented by WHO protocols to manage the prescription of antibiotics in therapeutic plans, strict cooperation among diagnostic laboratories, hospitals, and research institutes is strongly recommended to reduce the spread of these dangerous strains and prevent the emergence of serious infections in newborns. Considering this, we highlight the usefulness of molecular typing to better understand the distribution of resistant strains in clinical settings, especially where patients at risk, like neonates and children, are hospitalized.

4. Materials and Methods

4.1. Ethics Statement

The Institutional Review Board (IRB) of the IRCCS Bambino Gesù Children’s Hospital approved the study protocol (n.2156_OPBG_2020). As the data in this study were collected and analyzed retrospectively, the study did not infringe upon the rights or welfare of the patients and did not require their consent.

4.2. Study Design

This study was a retrospective investigation carried out from July 2016 to December 2019. A total of 9914 surveillance rectal swabs were performed for CPE screening from newborn patients hospitalized or admitted to the neonatal surgical unit (NSU), neonatal intensive therapy (NIT), the sub-intensive neonatal unit (SNU), and the cardiac intensive care unit (CICU) of Bambino Gesù Children’s Hospital in Rome (Italy), according to the active surveillance protocol issued by the hospital infection control committee. Wards considered in this study were those in which a possible transfer of patients frequently occurred during their hospital stay.
Strains were considered community-acquired when isolated from patients that were colonized at the time of their admission to the hospital. Otherwise, strains isolated from hospitalized patients were classified as hospital-acquired.
We collected demographic and clinical information about the enrolled patients from electronic medical records, including sex, age, geographical origin, the date of rectal swab collection, and department hospitalization (e.g., neonatal surgical unit, neonatal intensive therapy, sub-intensive neonatal unit, and cardiac intensive care unit). After isolation and identification, all CPE strains were characterized by antimicrobial susceptibility testing, detection of carbapenemase-encoding genes, and PBRT. Finally, one single strain of K. pneumoniae or E. coli representative for each PBRT profile was analyzed by MLST.

4.3. Microbiological Cultures and Antibiotic Susceptibility Testing

For cultural screening, each swab was inoculated on a set of two plates: MacConkey agar plate (bioMérieux, Craponne, France) with a 10 μg meropenem disk (Oxoid, Basingstoke, UK), according to EUCAST guidelines for the detection of resistance mechanisms (https://www.eucast.org/resistance_mechanisms/ (accessed on 21 February 2022)) and a CHROMID® CARBA plate (bioMérieux, Craponne, France), implemented in our routine laboratory practice.
Plates were incubated at 37 °C overnight. All the morphologically different colonies growing into the meropenem disk halo (zone diameter < 28 mm) and on the selective chromogenic medium were picked up and subcultured for purity onto a MacConkey agar plate (bioMérieux, Craponne, France) [54,55].
Isolated colonies were identified by using Matrix-Assisted Laser Desorption Ionization–Time-of-Flight Mass Spectrometry (MALDI-TOF, Bruker Daltonics, Bremen, Germany). Antimicrobial susceptibility testing was performed using the automated Vitek 2 (bioMérieux, Craponne, France) instrument. The following antimicrobial agents were tested with the automated system: amikacin (AMK), amoxicillin–clavulanic acid (AMC), cefepime (FEP), cefotaxime (CTX), ceftazidime (CAZ), ciprofloxacin (CIP), gentamicin (GEN), imipenem (IPM), meropenem (MEM), piperacillin–tazobactam (TZP), tigecycline (TGC), trimethoprim–sulfamethoxazole (SXT).
The MIC value of meropenem was confirmed with gradient test methods by MIC Test Strip (Liofilchem, Roseto degli Abruzzi, Italy) on Muller–Hinton agar (bioMérieux, Craponne, France), incubated at 37 °C overnight. The isolates were identified as resistant to carbapenems according to clinical breakpoints based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints tables. We adopted the updated EUCAST breakpoints tables (version 6.0 to version 9.0, from 2016 to 2019) (https://www.eucast.org/clinical_breakpoints/ (accessed on 21 February 2022)).

4.4. PCR-Based Methods for Carbapenemase Genes

The Xpert Carba-R molecular assay (Cepheid, Sunnyvale, CA, USA) was performed to confirm the presence of resistance genes to carbapenems. Briefly, subcultured isolated colonies were diluted in 0.45% saline to the turbidity of a 0.5 McFarland standard. Ten microliters of the suspension were inoculated into a 5 mL sample reagent vial and vortexed for 30 s. Finally, 1.7 mL of this suspension was transferred into an Xpert Carba-R cartridge using a disposable transfer pipette. The cartridge was loaded onto the GeneXpert system, and the assay was performed. The test, based on automated real-time PCR, is designed for rapid detection and differentiation of the blaKPC, blaNDM, blaVIM, blaOXA-48, and blaIMP-1 gene sequences associated with carbapenem-non-susceptible Gram-negative bacteria. The results were interpreted by the GeneXpert System from measured fluorescent signals.

4.5. Bacterial DNA Extraction and Plasmid Typing

Total DNA of isolated colonies that were positive for carbapenemase genes was extracted using an EZ1 DNA Tissue Kit (Qiagen, Hilden, Germany). Briefly, isolated colonies were diluted in 0.45% saline to the turbidity of 0.5 McFarland standard, and 200 µL of the suspension was transferred into a 2 mL vial. The vial and prefilled reagent cartridges were loaded onto the EZ1 Advanced XL (Qiagen, Hilden, Germany) instrument, and the protocol for automated purification of bacterial DNA, using magnetic particle technology, was started. DNA was eluted in a final volume of 100 µL and stored at −20 °C until use.
One µL of the extracted DNA was used for plasmid typing using a PCR-based replicon typing (PBRT) kit 2.0 (Diatheva, Fano, Italy). This novel PBRT assay, consisting of eight multiplex PCRs, is able to detect 30 different replicons of the main plasmid families in Enterobacterales [56]. This kit was used following the manufacturer’s instructions, including positive controls. Amplification products were resolved and visualized directly on a closed ready-to-use 2.2% agarose gel-cassette system (FlashGel-Lonza, Basel, Switzerland) using the 100 bp FlashGel DNA marker. The obtained fragments were compared to positive controls of each multiplex PCRs.

4.6. Multilocus Sequence Typing

Multilocus sequence typing (MLST) was performed to subtype K. pneumoniae and E. coli strains using housekeeping genes. For the former, the seven gene fragments (gapA, infB, mdh, pgi, phoE, rpoB, and tonB) were amplified by PCR and sequenced as described by protocol 2 of the Institute Pasteur Klebsiella MLST database (https://bigsdb.pasteur.fr/klebsiella/primers_used.html (accessed on 21 February 2022)). Instead, the seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA) to type E. coli strains were selected from the Enterobase MLST database (http://enterobase.warwick.ac.uk/species/index/ecoli (accessed on 28 February 2022). Primer sequences and reaction conditions were previously described by Wirth and colleagues (2006) [57].
All amplicons were sequenced using a BigDye Terminator v. 1.1 Cycle Sequencing kit on an ABI PRISM® 310 Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA). The alignment between sequences and the related reference was carried out using Unipro UGene version 38.0 software [58]. The allele numbers and the sequence types (STs) were determined through the corresponding MLST database. Finally, phylogenetic analyses of concatenated allelic variants were performed using Molecular Evolutionary Genetics Analysis (MEGA) software version 10.0. The evolutionary history was inferred using the Maximum Likelihood method and the General Time Reversible model. Initial trees for the heuristic search were obtained applying the Neighbor-Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. Branch quality was evaluated using a bootstrap test with 1000 replicates [59]. Graphical representation of both were obtained using Interactive Tree Of Lifes (iTOL v.6) software [60].

4.7. Whole-Genome Sequencing (WGS)

Whole-genome sequencing was performed in accordance with Lindsted et al. (2018) [61] for E. coli_104 strain, which revealed a new recA allele by MLST analysis. Briefly, genomic DNA concentration was determined using a Qubit dsDNA BR Assay Kit and a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, USA) in order to obtain a DNA input concentration between 100 and 500 ng. Library preparation was performed using an Illumina DNA Prep Library Kit (Illumina, Berlin, Germany) according to the manufacturer’s instructions. The reference strain of E. coli ATCC 25922 was used as sequencing quality control. Sequencing was performed using a MiSeq Reagent Micro Kit on an Illumina MiSeq desktop platform (Illumina Inc., San Diego, CA, USA) for ~20 h to produce paired-end sequences (2 × 300 base pair).
Raw Illumina reads were paired and assessed for sufficient coverage (≥Q30), and bases with low quality (<Q30) were discarded. Finally, the reads in FASTQ format were uploaded and submitted to the EnteroBase database (https://enterobase.warwick.ac.uk/species/index/ecoli (accessed on 21 February 2022)). High-throughput sequencing data were submitted to the Sequence Read Archive (SRA) (GeneBank accession number SUB10115582).
Futhermore, raw reads were analyzed using a bioinformatics tool of the Center for Genomic Epidemiology such as ResFinder 4.1 [62], PlasmidFinder 2.0 [63], pMLST-2.0 Server [63], and SerotypeFinder 2.0 Server [64].

5. Conclusions

Great attention should be given to high prevalence of CPE in NICUs and CICU due to the vulnerability of newborn patients. The molecular features of investigated strains showed their potential to be a serious threat to the health of neonates and infants. Our study contributed to increasing the knowledge concerning CPE circulation in Italian pediatric hospitals, and it highlighted the need to strengthen control measures to avoid their spread among wards. This is crucial to avoid complications for the treatment of neonatal infections. Moreover, introducing molecular typing methods in clinical routine procedures is becoming more and more necessary to monitor the spread of high-risk clones, to track their circulation, and to promptly identify emerging clones with advantageous ability to colonize clinical niches. We provided the evidence that PBRT can be commonly integrated within the diagnostic routine of a hospital in order to obtain important information about the epidemiology of circulating strains. Data concerning replicons harbored by MDROs in general, and CPE in our case study, represent an added value for a successful surveillance program. PBRT allows clinicians to prevent or monitor the spread of strains with potential ability to acquire and disseminate resistance genes through plasmids among wards. On the other hand, we used MLST because it enabled us to complete the description of the molecular profile of circulating clones, and the phylogenetic tree was also useful for comparing the novel identified ST with the well-known others. Moreover, typing housekeeping genes can be a very useful way to identify the dominance of a specific lineage responsible for an important clinical outbreak.

Author Contributions

Conceptualization, M.A. (Marilena Agosta), D.B., M.L.C.D.A., C.F.P., F.A., and P.B. (Paola Bernaschi); methodology, M.A. (Marilena Agosta), D.B., M.A. (Marta Argentieri), L.P. (Laura Pansani), L.M., A.S., C.D., L.P. (Lorenza Putignani), P.B. (Pietro Bagolan), B.D.I., L.D.C., A.D., F.A., and P.B. (Paola Bernaschi); formal analysis, M.A. (Marilena Agosta), D.B., M.A. (Marta Argentieri), L.P. (Laura Pansani), A.S., C.F.P., F.A., and P.B. (Paola Bernaschi); investigation, M.A. (Marilena Agosta), D.B., M.A. (Marta Argentieri), L.P. (Laura Pansani), A.S., M.L.C.D.A., C.D., L.P. (Lorenza Putignani), L.M., L.D.C., P.B. (Pietro Bagolan), B.D.I., A.D., M.M., C.F.P., F.A., and P.B. (Paola Bernaschi); writing—original draft preparation, M.A. (Marilena Agosta), D.B., M.L.C.D.A., F.A., and P.B. (Paola Bernaschi); writing—review and editing, M.A. (Marilena Agosta), D.B., M.L.C.D.A., P.B. (Pietro Bagolan), B.D.I., A.D., L.M., L.D.C., M.M., C.F.P., F.A., and P.B. (Paola Bernaschi); supervision, M.M., C.F.P., F.A., and P.B. (Paola Bernaschi); funding acquisition, M.M. and C.F.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Fano Ateneo (University of Urbino, Italy) and the Department of Diagnostic and Laboratory Medicine, Microbiology and Diagnostic Immunology Unit, Bambino Gesù Children’s Hospital (IRCCS, Rome, Italy).

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of the IRCCS Bambino Gesù Children’s Hospital, study protocol (n.2156_OPBG_2020).

Informed Consent Statement

As the data in this study were collected and analyzed retrospectively, the study did not infringe upon the rights or welfare of the patients and did not require their consent.

Data Availability Statement

All data are described within the text. The reads in FASTQ format of ST10555 were uploaded and submitted to the EnteroBase database (https://enterobase.warwick.ac.uk/species/index/ecoli (accessed on 21 February 2022)) and deposited in the Sequence Read Archive (SRA) (GeneBank accession number SUB10115582).

Conflicts of Interest

The authors declare no conflict of interests.

References

  1. Elshamy, A.A.; Aboshanab, K.M. A Review on Bacterial Resistance to Carbapenems: Epidemiology, Detection and Treatment Options. Futur. Sci. OA 2020, 6, FSO438. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  2. Tacconelli, E.; Carrara, E.; Savoldi, A.; Harbarth, S.; Mendelson, M.; Monnet, D.L.; Pulcini, C.; Kahlmeter, G.; Kluytmans, J.; Carmeli, Y.; et al. Discovery, Research, and Development of New Antibiotics: The WHO Priority List of Antibiotic-Resistant Bacteria and Tuberculosis. Lancet Infect. Dis. 2018, 18, 318–327. [Google Scholar] [CrossRef]
  3. Sherry, N.L.; Lane, C.R.; Kwong, J.C.; Schultz, M.; Sait, M.; Stevens, K.; Ballard, S.; Da Silva, A.G.; Seemann, T.; Gorrie, C.L.; et al. Genomics for Molecular Epidemiology and Detecting Transmission of Carbapenemase-Producing Enterobacterales in Victoria, Australia, 2012 to 2016. J. Clin. Microbiol. 2019, 57, e00573-19. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Logan, L.K.; Weinstein, R.A. The Epidemiology of Carbapenem-Resistant Enterobacteriaceae: The Impact and Evolution of a Global Menace. J. Infect. Dis. 2017, 215, S28–S36. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  5. Perez, F.; El Chakhtoura, N.G.; Papp-Wallace, K.M.; Wilson, B.M.; Bonomo, R.A. Treatment Options for Infections Caused by Carbapenem-Resistant Enterobacteriaceae. Expert Opin. Pharmacother. 2016, 17, 761–781. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  6. Räisänen, K.; Lyytikäinen, O.; Kauranen, J.; Tarkka, E.; Forsblom-Helander, B.; Grönroos, J.O.; Vuento, R.; Arifulla, D.; Sarvikivi, E.; Toura, S.; et al. Molecular Epidemiology of Carbapenemase-Producing Enterobacterales in Finland, 2012–2018. Eur. J. Clin. Microbiol. Infect. Dis. 2020, 39, 1651–1656. [Google Scholar] [CrossRef] [PubMed]
  7. Patel, G.; Bonomo, R.A. “Stormy Waters Ahead”: Global Emergence of Carbapenemases. Front. Microbiol. 2013, 4, 48. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  8. Munoz-Price, L.S.; Poirel, L.; Bonomo, R.A.; Schwaber, M.J.; Daikos, G.L.; Cormican, M.; Cornaglia, G.; Garau, J.; Gniadkowski, M.; Hayden, M.K.; et al. Clinical Epidemiology of the Global Expansion of Klebsiella Pneumoniae Carbapenemases. Lancet Infect. Dis. 2013, 13, 785–796. [Google Scholar] [CrossRef] [Green Version]
  9. Dortet, L.; Poirel, L.; Nordmann, P. Worldwide Dissemination of the NDM-Type Carbapenemases in Gram-Negative Bacteria. Biomed Res. Int. 2014, 2014, 1–12. [Google Scholar] [CrossRef] [Green Version]
  10. Carattoli, A. Plasmids in Gram Negatives: Molecular Typing of Resistance Plasmids. Int. J. Med. Microbiol. 2011, 301, 654–658. [Google Scholar] [CrossRef]
  11. Labi, A.K.; Bjerrum, S.; Enweronu-Laryea, C.C.; Ayibor, P.K.; Nielsen, K.L.; Marvig, R.L.; Newman, M.J.; Andersen, L.P.; Kurtzhals, J.A.L. High Carriage Rates of Multidrug-Resistant Gram- Negative Bacteria in Neonatal Intensive Care Units from Ghana. Open Forum Infect. Dis. 2020, 7, ofaa109. [Google Scholar] [CrossRef]
  12. Maida, C.M.; Bonura, C.; Geraci, D.M.; Graziano, G.; Carattoli, A.; Rizzo, A.; Torregrossa, M.V.; Vecchio, D.; Giuffrè, M. Outbreak of ST395 KPC-Producing Klebsiella Pneumoniae in a Neonatal Intensive Care Unit in Palermo, Italy. Infect. Control Hosp. Epidemiol. 2018, 39, 496–498. [Google Scholar] [CrossRef] [Green Version]
  13. Castagnola, E.; Tatarelli, P.; Mesini, A.; Baldelli, I.; La Masa, D.; Biassoni, R.; Bandettini, R. Epidemiology of Carbapenemase-Producing Enterobacteriaceae in a Pediatric Hospital in a Country with High Endemicity. J. Infect. Public Health 2019, 12, 270–274. [Google Scholar] [CrossRef]
  14. Ciccolini, M.; Donker, T.; Grundmann, H.; Bonten, M.J.M.; Woolhouse, M.E.J. Efficient Surveillance for Healthcare-Associated Infections Spreading between Hospitals. Proc. Natl. Acad. Sci. USA 2014, 111, 2271–2276. [Google Scholar] [CrossRef] [Green Version]
  15. Donker, T.; Wallinga, J.; Slack, R.; Grundmann, H. Hospital Networks and the Dispersal of Hospital-Acquired Pathogens by Patient Transfer. PLoS ONE 2012, 7, e35002. [Google Scholar] [CrossRef] [Green Version]
  16. Smith, D.L.; Dushoff, J.; Perencevich, E.N.; Harris, A.D.; Levin, S.A. Persistent Colonization and the Spread of Antibiotic Resistance in Nosocomial Pathogens: Resistance Is a Regional Problem. Proc. Natl. Acad. Sci. USA 2004, 101, 3709–3714. [Google Scholar] [CrossRef] [Green Version]
  17. Aguilera-Alonso, D.; Escosa-García, L.; Saavedra-Lozano, J.; Cercenado, E.; Baquero-Artigao, F. Carbapenem-Resistant Gram-Negative Bacterial Infections in Children. Antimicrob. Agents Chemother. 2020, 64, 1–19. [Google Scholar] [CrossRef]
  18. Tfifha, M.; Ferjani, A.; Mallouli, M.; Mlika, N.; Abroug, S.; Boukadida, J. Carriage of Multidrug-Resistant Bacteria among Pediatric Patients before and during Their Hospitalization in a Tertiary Pediatric Unit in Tunisia. Libyan J. Med. 2018, 13, 1419047. [Google Scholar] [CrossRef] [Green Version]
  19. Thuy, D.B.; Campbell, J.; Hoang Nhat, L.T.; VanMinh Hoang, N.; Van Hao, N.; Baker, S.; Geskus, R.B.; Thwaites, G.E.; Van Vinh Chau, N.; Louise Thwaites, C. Hospital-Acquired Colonization and Infections in a Vietnamese Intensive Care Unit. PLoS ONE 2018, 13, e0203600. [Google Scholar] [CrossRef]
  20. ECDC. Surveillance of Antimicrobial Resistance in Europe 2018; ECDC: Solna, Sweden, 2018. [Google Scholar]
  21. Public Health England Health Protection Report: Latest Surveillance Reports. Available online: https://www.gov.uk/government/collections/health-protection-report-latest-infection-reports (accessed on 2 July 2021).
  22. CDC Carbapenem-Resistant Enterobacterales (CRE). Available online: https://www.cdc.gov/hai/organisms/cre/index.html (accessed on 2 July 2021).
  23. Yin, L.; He, L.; Miao, J.; Yang, W.; Wang, X.; Ma, J.; Wu, N.; Cao, Y.; Wang, L.; Lu, G.; et al. Active Surveillance and Appropriate Patient Placement in Contact Isolation Dramatically Decreased Carbapenem-Resistant Enterobacterales Infection and Colonization in Paediatric Patients in China. J. Hosp. Infect. 2020, 105, 486–494. [Google Scholar] [CrossRef]
  24. Simon, A.; Tenenbaum, T. Surveillance of Multidrug-Resistant Gram-Negative Pathogens in High-Risk Neonates-Does It Make a Difference? Pediatr. Infect. Dis. J. 2013, 32, 407–409. [Google Scholar] [CrossRef] [PubMed]
  25. Gagliotti, C.; Ciccarese, V.; Sarti, M.; Giordani, S.; Barozzi, A.; Braglia, C.; Gallerani, C.; Gargiulo, R.; Lenzotti, G.; Manzi, O.; et al. Active Surveillance for Asymptomatic Carriers of Carbapenemase-Producing Klebsiella Pneumoniae in a Hospital Setting. J. Hosp. Infect. 2013, 83, 330–332. [Google Scholar] [CrossRef] [PubMed]
  26. Meredith, H.R.; Kularatna, S.; Nagaro, K.; Nagahawatte, A.; Bodinayake, C.; Kurukulasooriya, R.; Wijesingha, N.; Harden, L.B.; Piyasiri, B.; Hammouda, A.; et al. Colonization with Multidrug-Resistant Enterobacteriaceae among Infants: An Observational Study in Southern Sri Lanka. Antimicrob. Resist. Infect. Control 2021, 10, 72. [Google Scholar] [CrossRef] [PubMed]
  27. Montagnani, C.; Prato, M.; Scolfaro, C.; Colombo, S.; Esposito, S.; Tagliabue, C.; Lo Vecchio, A.; Bruzzese, E.; Loy, A.; Cursi, L.; et al. Carbapenem-Resistant Enterobacteriaceae Infections in Children: An Italian Retrospective Multicenter Study. Pediatr. Infect. Dis. J. 2016, 35, 862–868. [Google Scholar] [CrossRef]
  28. Giuffrè, M.; Bonura, C.; Geraci, D.M.; Saporito, L.; Catalano, R.; Di Noto, S.; Nociforo, F.; Corsello, G.; Mammina, C. Successful Control of an Outbreak of Colonization by Klebsiella Pneumoniae Carbapenemase-Producing K. Pneumoniae Sequence Type 258 in a Neonatal Intensive Care Unit, Italy. J. Hosp. Infect. 2013, 85, 233–236. [Google Scholar] [CrossRef] [Green Version]
  29. Geraci, D.M.; Bonura, C.; Giuffrè, M.; Saporito, L.; Graziano, G.; Aleo, A.; Fasciana, T.; Di Bernardo, F.; Stampone, T.; Palma, D.M.; et al. Is the Monoclonal Spread of the ST258, KPC-3-Producing Clone Being Replaced in Southern Italy by the Dissemination of Multiple Clones of Carbapenem-Nonsusceptible, KPC-3-Producing Klebsiella Pneumoniae? Clin. Microbiol. Infect. 2015, 21, e15–e17. [Google Scholar] [CrossRef] [Green Version]
  30. Mammina, C.; Di Carlo, P.; Cipolla, D.; Giuffrè, M.; Casuccio, A.; Di Gaetano, V.; Plano, M.R.A.; D’Angelo, E.; Titone, L.; Corsello, G. Surveillance of Multidrug-Resistant Gram-Negative Bacilli in a Neonatal Intensive Care Unit: Prominent Role of Cross Transmission. Am. J. Infect. Control 2007, 35, 222–230. [Google Scholar] [CrossRef]
  31. Singh, N.P.; Choudhury, D.D.; Gupta, K.; Rai, S.; Batra, P.; Manchanda, V.; Saha, R.; Kaur, I.R. Predictors for Gut Colonization of Carbapenem-Resistant Enterobacteriaceae in Neonates in a Neonatal Intensive Care Unit. Am. J. Infect. Control 2018, 46, e31–e35. [Google Scholar] [CrossRef]
  32. Han, R.; Shi, Q.; Wu, S.; Yin, D.; Peng, M.; Dong, D.; Zheng, Y.; Guo, Y.; Zhang, R.; Hu, F.; et al. Dissemination of Carbapenemases (KPC, NDM, OXA-48, IMP, and VIM) Among Carbapenem-Resistant Enterobacteriaceae Isolated From Adult and Children Patients in China. Front. Cell. Infect. Microbiol. 2020, 10, 314. [Google Scholar] [CrossRef]
  33. Almeida, T.L.; Mendo, T.; Costa, R.; Novais, C.; Marçal, M.; Martins, F.; Tuna, M. Carbapenemase-Producing Enterobacteriaceae (CPE) Newborn Colonization in a Portuguese Neonatal Intensive Care Unit (NICU): Epidemiology and Infection Prevention and Control Measures. Infect. Dis. Rep. 2021, 13, 411–417. [Google Scholar] [CrossRef]
  34. Magiorakos, A.P.; Srinivasan, A.; Carey, R.B.; Carmeli, Y.; Falagas, M.E.; Giske, C.G.; Harbarth, S.; Hindler, J.F.; Kahlmeter, G.; Olsson-Liljequist, B.; et al. Multidrug-Resistant, Extensively Drug-Resistant and Pandrug-Resistant Bacteria: An International Expert Proposal for Interim Standard Definitions for Acquired Resistance. Clin. Microbiol. Infect. 2012, 18, 268–281. [Google Scholar] [CrossRef] [Green Version]
  35. Sader, H.S.; Castanheira, M.; Farrell, D.J.; Flamm, R.K.; Mendes, R.E.; Jones, R.N. Tigecycline Antimicrobial Activity Tested against Clinical Bacteria from Latin American Medical Centres: Results from SENTRY Antimicrobial Surveillance Program (2011–2014). Int. J. Antimicrob. Agents 2016, 48, 144–150. [Google Scholar] [CrossRef]
  36. Zhang, R.; Dong, N.; Huang, Y.; Zhou, H.; Xie, M.; Chan, E.W.C.; Hu, Y.; Cai, J.; Chen, S. Evolution of Tigecycline- and Colistin-Resistant CRKP (Carbapenem-Resistant Klebsiella Pneumoniae) in Vivo and Its Persistence in the GI Tract Article. Emerg. Microbes Infect. 2018, 7, 1–11. [Google Scholar] [CrossRef] [Green Version]
  37. Ojdana, D.; Gutowska, A.; Sacha, P.; Majewski, P.; Wieczorek, P.; Tryniszewska, E. Activity of Ceftazidime-Avibactam Alone and in Combination with Ertapenem, Fosfomycin, and Tigecycline against Carbapenemase-Producing Klebsiella Pneumoniae. Microb. Drug Resist. 2019, 25, 1357–1364. [Google Scholar] [CrossRef]
  38. Folgori, L.; Bielicki, J. Future Challenges in Pediatric and Neonatal Sepsis: Emerging Pathogens and Antimicrobial Resistance. J. Pediatr. Intensiv. Care 2019, 08, 017–024. [Google Scholar] [CrossRef]
  39. Nordmann, P.; Naas, T.; Poirel, L. Global Spread of Carbapenemase Producing Enterobacteriaceae. Emerg. Infect. Dis. 2011, 17, 1791–1798. [Google Scholar] [CrossRef]
  40. ECDC. European Centre for Disease Prevention and Control Regional Outbreak of New Delhi Metallo-Beta-Lactamase-Producing Carbapenem-Resistant Enterobacteriaceae, Italy, 2018–2019; ECDC: Solna, Sweden, 2019; pp. 1–7. [Google Scholar]
  41. Wu, W.; Feng, Y.; Tang, G.; Qiao, F.; McNally, A.; Zong, Z. NDM Metallo-β-Lactamases and Their Bacterial Producers in Health Care Settings. Clin. Microbiol. Rev. 2019, 32, e00115-18. [Google Scholar] [CrossRef] [Green Version]
  42. Carattoli, A.; Villa, L.; Poirel, L.; Bonnin, R.A.; Nordmann, P. Evolution of IncA/C Bla CMY-2-Carrying Plasmids by Acquisition of the BlaNDM-1 Carbapenemase Gene. Antimicrob. Agents Chemother. 2012, 56, 783–786. [Google Scholar] [CrossRef] [Green Version]
  43. Hancock, S.J.; Phan, M.D.; Peters, K.M.; Forde, B.M.; Chong, T.M.; Yin, W.F.; Chan, K.G.; Paterson, D.L.; Walsh, T.R.; Beatson, S.A.; et al. Identification of IncA/C Plasmid Replication and Maintenance Genes and Development of a Plasmid Multilocus Sequence Typing Scheme. Antimicrob. Agents Chemother. 2017, 61, e01740-16. [Google Scholar] [CrossRef] [Green Version]
  44. Welch, T.J.; Fricke, W.F.; McDermott, P.F.; White, D.G.; Rosso, M.L.; Rasko, D.A.; Mammel, M.K.; Eppinger, M.; Rosovitz, M.J.; Wagner, D.; et al. Multiple Antimicrobial Resistance in Plague: An Emerging Public Health Risk. PLoS ONE 2007, 2, e309. [Google Scholar] [CrossRef] [Green Version]
  45. Winokur, P.L.; Vonstein, D.L.; Hoffman, L.J.; Uhlenhopp, E.K.; Doern, G.V. Evidence for Transfer of CMY-2 AmpC β-Lactamase Plasmids between Escherichia Coli and Salmonella Isolates from Food Animals and Humans. Antimicrob. Agents Chemother. 2001, 45, 2716–2722. [Google Scholar] [CrossRef] [Green Version]
  46. Villa, L.; García-Fernández, A.; Fortini, D.; Carattoli, A. Replicon Sequence Typing of IncF Plasmids Carrying Virulence and Resistance Determinants. J. Antimicrob. Chemother. 2010, 65, 2518–2529. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  47. Barbadoro, P.; Bencardino, D.; Carloni, E.; Omiccioli, E.; Ponzio, E.; Micheletti, R.; Acquaviva, G.; Luciani, A.; Masucci, A.; Pocognoli, A.; et al. Carriage of Carbapenem-Resistant Enterobacterales in Adult Patients Admitted to a University Hospital in Italy. Antibiotics 2021, 10, 61. [Google Scholar] [CrossRef] [PubMed]
  48. Magi, G.; Tontarelli, F.; Caucci, S.; Di Sante, L.; Brenciani, A.; Morroni, G.; Giovanetti, E.; Menzo, S.; Mingoia, M. High Prevalence of Carbapenem-Resistant Klebsiella Pneumoniae ST307 Recovered from Fecal Samples in an Italian Hospital. Fut. Microbiol. 2021, 16, 703–711. [Google Scholar] [CrossRef]
  49. Villa, L.; Feudi, C.; Fortini, D.; Brisse, S.; Passet, V.; Bonura, C.; Endimiani, A.; Mammina, C.; Ocampo, A.M.; Jimenez, J.N.; et al. Diversity, Virulence, and Antimicrobial Resistance of the KPCproducing Klebsiella Pneumoniae ST307 Clone. Microb. Genom. 2017, 3, e000110. [Google Scholar] [CrossRef] [PubMed]
  50. Loconsole, D.; Accogli, M.; De Robertis, A.L.; Capozzi, L.; Bianco, A.; Morea, A.; Mallamaci, R.; Quarto, M.; Parisi, A.; Chironna, M. Emerging High-Risk ST101 and ST307 Carbapenem-Resistant Klebsiella Pneumoniae Clones from Bloodstream Infections in Southern Italy. Ann. Clin. Microbiol. Antimicrob. 2020, 19, 24. [Google Scholar] [CrossRef] [PubMed]
  51. Mammina, C.; Palma, D.M.; Bonura, C.; Plano, M.R.A.; Monastero, R.; Sodano, C.; Calà, C.; Tetamo, R. Outbreak of Infection with Klebsiella Pneumoniae Sequence Type 258 Producing Klebsiella Pneumoniae Carbapenemase 3 in an Intensive Care Unit in Italy. J. Clin. Microbiol. 2010, 48, 1506–1507. [Google Scholar] [CrossRef] [Green Version]
  52. Bonura, C.; Giuffrè, M.; Aleo, A.; Fasciana, T.; Di Bernardo, F.; Stampone, T.; Giammanco, A.; Palma, D.M.; Mammina, C.; Geraci, D.M.; et al. An Update of the Evolving Epidemic of BlaKPC Carrying Klebsiella Pneumoniae in Sicily, Italy, 2014: Emergence of Multiple Non-ST258 Clones. PLoS ONE 2015, 10, e0132936. [Google Scholar] [CrossRef] [Green Version]
  53. Forde, B.M.; Roberts, L.W.; Phan, M.D.; Peters, K.M.; Fleming, B.A.; Russell, C.W.; Lenherr, S.M.; Myers, J.B.; Barker, A.P.; Fisher, M.A.; et al. Population Dynamics of an Escherichia Coli ST131 Lineage during Recurrent Urinary Tract Infection. Nat. Commun. 2019, 10, 3643. [Google Scholar] [CrossRef] [Green Version]
  54. Arena, F.; Di Pilato, V.; Vannetti, F.; Fabbri, L.; Antonelli, A.; Coppi, M.; Pupillo, R.; Macchi, C.; Rossolini, G.M. Population Structure of Kpc Carbapenemase-Producing Klebsiella Pneumoniae in a Long-Term Acute-Care Rehabilitation Facility: Identification of a New Lineage of Clonal Group 101, Associated with Local Hyperendemicity. Microb. Genomics 2020, 6, e000308. [Google Scholar] [CrossRef]
  55. Cortegiani, A.; Russotto, V.; Graziano, G.; Geraci, D.; Saporito, L.; Cocorullo, G.; Raineri, S.M.; Mammina, C.; Giarratano, A. Use of Cepheid Xpert Carba-R® for Rapid Detection of Carbapenemase-Producing Bacteria in Abdominal Septic Patients Admitted to Intensive Care Unit. PLoS ONE 2016, 11, e0160643. [Google Scholar] [CrossRef]
  56. Carloni, E.; Andreoni, F.; Omiccioli, E.; Villa, L.; Magnani, M.; Carattoli, A. Comparative Analysis of the Standard PCR-Based Replicon Typing (PBRT) with the Commercial PBRT-KIT. Plasmid 2017, 90, 10–14. [Google Scholar] [CrossRef]
  57. Wirth, T.; Falush, D.; Lan, R.; Colles, F.; Mensa, P.; Wieler, L.H.; Karch, H.; Reeves, P.R.; Maiden, M.C.J.; Ochman, H.; et al. Sex and Virulence in Escherichia Coli: An Evolutionary Perspective. Mol. Microbiol. 2006, 60, 1136–1151. [Google Scholar] [CrossRef] [Green Version]
  58. Okonechnikov, K.; Golosova, O.; Fursov, M.; Varlamov, A.; Vaskin, Y.; Efremov, I.; German Grehov, O.G.; Kandrov, D.; Rasputin, K.; Syabro, M.; et al. Unipro UGENE: A Unified Bioinformatics Toolkit. Bioinformatics 2012, 28, 1166–1167. [Google Scholar] [CrossRef] [Green Version]
  59. Kumar, S.; Stecher, G.; Li, M.; Knyaz, C.; Tamura, K. MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol. Biol. Evol. 2018, 35, 1547–1549. [Google Scholar] [CrossRef]
  60. Letunic, I.; Bork, P. Interactive Tree of Life (ITOL) v5: An Online Tool for Phylogenetic Tree Display and Annotation. Nucleic Acids Res. 2021, 49, W293–W296. [Google Scholar] [CrossRef]
  61. Lindstedt, B.-A.; Finton, M.D.; Porcellato, D.; Brandal, L.T. High Frequency of Hybrid Escherichia Coli Strains with Combined Intestinal Pathogenic Escherichia Coli (IPEC) and Extraintestinal Pathogenic Escherichia Coli (ExPEC) Virulence Factors Isolated from Human Faecal Samples. BMC Infect. Dis. 2018, 18, 10–14. [Google Scholar] [CrossRef]
  62. Bortolaia, V.; Kaas, R.S.; Ruppe, E.; Roberts, M.C.; Schwarz, S.; Cattoir, V.; Philippon, A.; Allesoe, R.L.; Rebelo, A.R.; Florensa, A.F.; et al. ResFinder 4.0 for Predictions of Phenotypes from Genotypes. J. Antimicrob. Chemother. 2020, 75, 3491–3500. [Google Scholar] [CrossRef]
  63. Carattoli, A.; Zankari, E.; García-Fernández, A.; Larsen, M.V.; Lund, O.; Villa, L.; Aarestrup, F.M.; Hasman, H. In Silico Detection and Typing of Plasmids Using PlasmidFinder and Plasmid Multilocus Sequence Typing. Antimicrob. Agents Chemother. 2014, 58, 3895–3903. [Google Scholar] [CrossRef] [Green Version]
  64. Joensen, K.G.; Tetzschner, A.M.M.; Iguchi, A.; Aarestrup, F.M.; Scheutz, F. Rapid and Easy in Silico Serotyping of Escherichia Coli Isolates by Use of Whole-Genome Sequencing Data. J. Clin. Microbiol. 2015, 53, 2410–2426. [Google Scholar] [CrossRef] [Green Version]
Antibiotics 11 00431 g001 550
Figure 1. Distribution of PBRT patterns among CPE strains. Forty-nine PBRT profiles were distributed among 122 out of 124 CPE strains. All of these PBRT patterns were associated with a single species with the exception of replicon A/C (E. coli, E. cloacae, M. morganii, K. aerogenes, and K. oxytoca) and IncFIB KQ, IncFIIK (K. pneumoniae and C. freundii).
Figure 1. Distribution of PBRT patterns among CPE strains. Forty-nine PBRT profiles were distributed among 122 out of 124 CPE strains. All of these PBRT patterns were associated with a single species with the exception of replicon A/C (E. coli, E. cloacae, M. morganii, K. aerogenes, and K. oxytoca) and IncFIB KQ, IncFIIK (K. pneumoniae and C. freundii).
Antibiotics 11 00431 g001
Antibiotics 11 00431 g002 550
Figure 2. MLST-based phylogenetic trees of K. pneumoniae and E. coli strains. Trees of concatenated nucleotide sequences of seven housekeeping genes of K. pneumoniae (A) and E. coli (B) were obtained using the Maximum Likelihood method and the General Time Reversible model by MEGA software (version 10.0) with bootstrap percentages retrieved in 1000 replications. Graphical representation of both were obtained using Interactive Tree of Lifes (iTOL v.6) software. Scale bars indicate the number of nucleotide substitutions per site. For each strain, the name was reported as Kp for K. pneumoniae and Ec for E. coli. STs and replicon profiles were also indicated for each strain.
Figure 2. MLST-based phylogenetic trees of K. pneumoniae and E. coli strains. Trees of concatenated nucleotide sequences of seven housekeeping genes of K. pneumoniae (A) and E. coli (B) were obtained using the Maximum Likelihood method and the General Time Reversible model by MEGA software (version 10.0) with bootstrap percentages retrieved in 1000 replications. Graphical representation of both were obtained using Interactive Tree of Lifes (iTOL v.6) software. Scale bars indicate the number of nucleotide substitutions per site. For each strain, the name was reported as Kp for K. pneumoniae and Ec for E. coli. STs and replicon profiles were also indicated for each strain.
Antibiotics 11 00431 g002
Table
Table 1. Genotypic and phenotypic profile of CPE strains isolated from rectal swabs.
Table 1. Genotypic and phenotypic profile of CPE strains isolated from rectal swabs.
Strains aAge bSexOrigin cCAC or HAC dClinical Ward eDateSpeciesAntibiotic Resistance Pattern fGenesPBRT gST
84-A5 mthsMRussiaCACNSU6/9/17K. pneumoniaeAMK-AMC-FEP-CTX-CAZ-CIP-GEN-IPM-TZP-SXT-MEMblaKPC + blaNDM(1) A/C, R, FIIK, FIB KQ
11624 daysMItalyHACNIT8/7/17395
1276 daysFNSU8/14/17
12314 daysM8/14/17
1452 mthsMSNU10/2/17
17219 daysF11/6/17
12211 daysFNIT8/14/17AMK-AMC-FEP-CTX-CAZ-CIP-GEN-IPM-TZP-MEM
126-D1 mthMNSU8/14/17AMK-AMC-FEP-CTX-CAZ-CIP-GEN-IPM-TZP-TGC-SXT-MEM
1582 mthsFItalyCACNIT10/14/17K. pneumoniaeAMC-CTX-CAZ-IPM-TZP-MEMblaKPC(2) FIB KQ, FIIK1164
1554 mthsHACNSU10/9/17C. freundii
1592 mthsMItalyHACSNU10/16/17K. pneumoniaeAMK-AMC-FEP-CTX-CAZ-GEN-IPM-TZP-MEMblaNDM(3) A/C, FIB KN17
168a-L13 daysNSU
16020 daysAMK-AMC-FEP-CTX-CAZ-GEN-IPM-TZP-SXT-MEM
1893 mthsFItalyHACNIT12/11/17K. pneumoniaeAMK-AMC-FEP-CTX-CAZ-GEN-IPM-TZP-MEMblaNDM(4) FIB KQ, R, A/C54
167-G4 mthsFItalyHACNSU10/16/17E. coliAMK-AMC-FEP-CTX-CAZ-CIP-GEN-IPM-TZP-SXT-MEMblaNDM(5) FII, A/C, FIA
138-D2 mthsM9/18/17AMK-AMC-FEP-CTX-CAZ-CIP-GEN-IPM-TZP-MEM
187-O2 mthsF12/4/17
18815 daysFSNU12/11/17131
1914 mthsMNSU12/11/17
204-R1 mthM2/5/18
207-Q2 mthsM2/9/18
211-T17 daysF2/19/18
21418 daysF2/26/18
2151 mthM2/26/18
2162 mthsM2/26/18
218-U2 mthsF2/26/18
2223 mthsMNIT3/5/18
2271 mthMSNU3/12/18
226-Z2 mthsFNSU3/12/18
228-S1 yearM3/12/18
2326 daysM3/19/18
240-Y1 mthM4/16/18
274-Z7 mthsF8/8/18
163-M1 mthMItalyHACNSU10/16/17E. coliAMK-AMC-FEP-CTX-CAZ-GEN-IPM-TZP-MEMblaNDM(6) FII, A/C, FIB
168b-L13 daysMNSU10/16/17
169-I1 mthFNSU10/16/17
2101 mthsMNIT2/19/18
2456 mthsFNIT4/23/1874
25029 daysMNSU5/7/18
2612 mthsFCACNIT6/30/18AMC-FEP-CTX-CAZ-IPM-TZP-SXTblaVIM
2754 mthsMHACNSU8/20/18
26614 daysMSNU7/25/18AMC-FEP-CTX-CAZ-IPM-TZP-SXT-MEMblaVIM
3361 yearMS. LeoneNSU7/15/19blaNDM
199-P3 mthsMItalyNIT1/10/18AMK-AMC-FEP-CTX-CAZ-TZPblaNDM
2475 mthsFRomaniaCACNIT4/28/18K. pneumoniaeAMC-FEP-CTX-CAZ-CIP-GEN-IPM-TZP-SXTblaOXA-48(7) FIB KQ, FIB KN, FII K, L307
2851 mthMItalyCACNIT9/28/18K. pneumoniaeAMK-AMC-FEP-CTX-CAZ-CIP-GEN-IPM-TZP-MEMblaKPC(8) FIB KN, FII K, R101
161 mthFRomaniaCACNSU9/22/16K. pneumoniaeAMK-AMC-FEP-CTX-CAZ-CIP-GEN-IPM-TZP-TGC-MEMblaOXA-48(9) R, L11
831 mthsMItalyHACNSU6/5/17K. pneumoniaeAMK-AMC-FEP-CTX-CAZ-CIP-IPM-TZP-MEMblaKPC(10) FIB KN, FII K, X3512
90-A5 mthsMRussiaHACNSU6/19/17E. coliAMK-AMC-FEP-CTX-CAZ-CIP-GEN-IPM-TZP-SXT-MEMblaNDM(11) FII, FII K, A/C, FIA, FIB131
104-B5 mthsMItalyHACNSU7/18/17E. coliAMK-AMC-FEP-CTX-CAZ-CIP-GEN-IPM-TZP-SXT-MEMblaNDM(12) A/C10555
1291 mthM8/14/17K. aerogenesAMK-AMC-FEP-CTX-CAZ-GEN-IPM-TZP-MEM
13015 daysM8/18/17E. coli
131-C1 mthF8/21/17
1836 mthsM11/27/17K. aerogenesblaKPC+blaNDM
2297 mthsM3/12/18K. oxytocaAMC-CTX-CAZ-IPM-TZP-MEMblaNDM
221-K1 yearMRussia3/2/18M. morganiiAMK-AMC-CTX-CAZ-GEN-IPM-MEMblaNDM+blaVIM
2464 mthsMItaly4/26/18E. cloacaeAMK-AMC-FEP-CTX-CAZ-GEN-TZP-MEMblaNDM
1061 mthMItalyHACNSU7/24/17K. pneumoniaeAMK-AMC-FEP-CTX-CAZ-CIP-GEN-IPM-TZP-SXT-MEMblaKPC+blaNDM(13) A/C, R, FII K395
117-C1 mthF8/7/17
12411 daysFItalyHACNSU8/14/17K. pneumoniaeAMC-CTX-CAZ-IPM-TZP-MEMblaKPC(14) FIB KQ, FIB KN, FII K17
1255 days8/14/17
103-B5 mthsMItalyHACNSU7/17/17K. pneumoniaeAMC-FEP-CTX-CAZ-CIP-GEN-IPM-TZP-SXTblaKPC(15) X2307
134-E1 mthFBurundiCACNSU8/30/17E. coliAMC-FEP-CTX-CAZ-CIP-GEN-IPM-TZP-SXT-MEMblaNDM(16) FII, X3, FIA, FIB617
137-F1 mth9/4/17
166-N1 mthItalyHAC10/16/17
1745 mthsLibya11/13/17
1393 mthsMItalyHACNSU9/18/17K. pneumoniaeAMK-AMC-FEP-CTX-CAZ-CIP-GEN-IPM-TZP-SXT-MEMblaKPC+blaNDM(17) FIB KQ, FIIK, R, A/C, FIA395
144-H4 mthsF10/2/17
14113 daysF10/2/17
143-G4 mthsF10/2/17
162-M1 mthM10/16/17
171-N1 mthF10/23/17
209-S11mthsM2/12/18
146-I16 daysFItalyHACNSU10/2/17K. pneumoniaeAMK-AMC-FEP-CTX-CAZ-GEN-IPM-TZP-MEMblaKPC+blaNDM(18) FIB KN, FIIK, R, A/C17
153-F3 mthsFBurundiHACNSU10/9/17K. pneumoniaeAMC-FEP-CTX-CAZ-CIP-IPM-TZP-SXT-MEMblaNDM(19) FIB-M, HIB-M, R, X3323
154-E3 mths10/9/17
161-O17 daysFItalyHACNSU10/16/17K. pneumoniaeAMK-AMC-FEP-CTX-CAZ-GEN-IPM-TZP-TGC-MEMblaNDM(20) FIB KN, A/C, N17
177-P2 mthsMItalyHACNSU11/20/17K. pneumoniaeAMK-AMC-FEP-CTX-CAZ-GEN-IPM-TZP-MEMblaNDM(21) FIA, A/C, R, FIB KQ54
17826 days11/20/17blaKPC+blaNDM
181-Q13 days11/27/17blaNDM
18219 days11/27/17blaNDM
190-H6 mthsFItalyHACNSU12/11/17E. coliAMK-AMC-FEP-CTX-CAZ-GEN-TZP-SXT-MEMblaNDM(22) FII, A/C80
195-R16 daysMItalyHACNSU1/3/18K. pneumoniaeAMK-AMC-FEP-CTX-CAZ-CIP-GEN-IPM-TZP-TGC-SXT-MEMblaKPC + blaNDM(23) FIIK, FIIS, P1395
2009 mthsMItalyCACNSU1/17/18K. pneumoniaeAMC-FEP-CTX-CAZ-CIP-GEN-IPM-TZP-SXT-MEMblaKPC(24) K, FIB KN, X2, P1307
2892 mthsHACCICU10/15/18
224-V6 mthsMItalyHACCICU3/11/18E. coliAMC-FEP-CTX-CAZ-IPM-TZP-SXT-MEMblaVIM(25) FII, FIB, M162
225-V6 mthsMItalyHACCICU3/11/18K. pneumoniaeAMC-FEP-CTX-CAZ-IPM-TZP-SXT-MEMblaNDM + blaVIM(26) FIB KN, FIA, M466
25720 daysFCICU6/13/18AMC-FEP-CTX-CAZ-GEN-IPM-TZP-SXT-MEMblaNDM
230-U3 mthsFItalyHACNSU3/12/18K. pneumoniaeAMK-AMC-FEP-CTX-CAZ-CIP-GEN-IPM-TZP-SXT-MEMblaNDM(27) R, FII K, FIB KN, A/C, FIA231
231-T1 mthFItalyCACNSU3/18/18K. pneumoniaeAMK-AMC-FEP-CTX-CAZ-GEN-IPM-TZP-MEMblaNDM(28) A/C, FIA35
237-Y23 daysMHAC4/9/18
2782 mthsFC.A.R.CACNSU9/11/18E. coliAMK-AMC-FEP-CTX-CAZ-CIP-GEN-IPM-TZP-SXT-MEMblaNDM(29) I1α, FIA, FII167
341-X3 mthsFUkraineCACNSU8/4/19E. coliAMK-AMC-FEP-CTX-CAZ-CIP-GEN-IPM-TZP-SXT-MEMblaNDM(30) I1α, N, A/C, FII69
3127 mthsFGeorgiaCACNSU3/9/19E. coliAMC-FEP-CTX-CAZ-IPM-TZP-SXT-MEMblaNDM(31) FIB, T, HIB-M, FIB-M, FII131
369-W4 mthsFItalyHACNSU10/29/19K. pneumoniaeAMK-AMC-FEP-CTX-CAZ-GEN-IPM-TZP-MEMblaNDM(32) M, FIA, A/C, HIB-M, FIB-M160
2883 mthsFItalyCACNSU10/11/18K. pneumoniaeAMK-AMC-FEP-CTX-CAZ-CIP-GEN-IPM-TZP-MEMblaKPC(33) FIA, R, FIIK, FIB KN101
2982 mthsM12/3/18
3086 mthsF2/21/19
3933 mthsMIraqHACCICU1/28/20
2353 mthsFItalyCACNSU4/4/18K. pneumoniaeAMC-CTX-CAZ-CIP-IPM-TZP-MEMblaKPC(34) FIA, FIIK, FIB KQ, HIB-M, FIB-M307
3046 daysFItalyCACNSU1/29/19K. pneumoniaeAMK-AMC-FEP-CTX-CAZ-CIP-GEN-IPM-TZP-MEMblaKPC(35) M, N, FIA, R, FIIK101
3332 mthsFItalyHACNIT7/1/19K. pneumoniaeAMK-AMC-FEP-CTX-CAZ-CIP-GEN-IPM-TZP-SXT-MEMblaKPC + blaNDM(36) M, FIA, FIIK, FIB KN, FIB KQ307
342-X3 mthsFUkraineCACNSU8/4/19K. pneumoniaeAMC-FEP-CTX-CAZ-CIP-GEN-IPM-TZP-SXT-MEMblaNDM(37) N, A/C, FIIK, FII1412
34522 daysMItalyHACNSU8/19/19K. pneumoniaeAMC-FEP-CTX-CAZ-CIP-GEN-IPM-TZP-SXT-MEMblaNDM(38) A/C, FIIK, FII1412
350-W3 mthsFItalyHACNSU9/2/19E. coliAMK-AMC-FEP-CTX-CAZ-CIP-GEN-IPM-TZP-SXT-MEMblaNDM(39) N, A/C, FII69
1422 mthsFBurundiHACNSU10/2/17E. cloacaeAMC-FEP-CTX-CAZ-GEN-IPM-TZP-MEMblaKPC + blaNDM(40) X3, N
1651 mthFItalyHACNSU10/16/17E. cloacaeAMC-FEP-CTX-CAZ-IPM-TZP-MEMblaNDM(41) FII, T, X3, FIA, FIB
1795 mthsFLibyaHACNSU11/20/17C. koseriAMC-FEP-CTX-CAZ-IPM-TZP-MEMblaNDM(42) FIB, X3
208-K1 yearMRussiaHACNSU2/11/18K. oxytocaAMK-AMC-CTX-CAZ-GEN-IPM-TZP-MEMblaNDM(43) A/C, FIIK, FIB KN
23415 daysMItalyHACNSU4/3/18K. oxytocaAMK-AMC-FEP-CTX-CAZ-GEN-IPM-TZP-MEMblaKPC + blaNDM(44) A/C, U
2414 mths4/16/18blaNDM
2422 mths4/16/18
2431 mth4/16/18
24819 days4/30/18
3741 mthFItalyHACNSU11/11/19C. freundiiAMC-FEP-CTX-CAZ-GEN-IPM-TZP-MEMblaVIM(45) FII, FIB KN, M, HI2
3701 mthMItalyHACNSU11/4/19C. freundiiAMC-FEP-CTX-CAZ-IPM-TZP-MEMblaVIM(46) FIB KN, FII, HI2
3752 mthsF11/11/19
3883 mthsM12/10/19
37718 daysFItalyCACNIT11/12/19S. marcescensAMK-AMC-FEP-CTX-IPM-TZP-MEMblaOXA-48(47) L
3811 mthMItalyHACNSU11/18/19C. freundiiAMK-AMC-FEP-CTX-CAZ-GEN-IPM-TZP-MEMblaNDM(48) A/C, FIB KN, HIB-M, FII, FIB-M
3371 yearMItalyHACNIT7/15/19E. cloacaeAMC-FEP-CTX-CAZ-CIP-GEN-IPM-TZP-SXT-MEMblaVIM(49) HI2
38928 daysNSU12/16/19
3732 daysMItalyCACSNU11/7/19E. cloacaeAMC-FEP-CTX-CAZ-GEN-IPM-TZP-SXT-MEMblaVIMUT
48 mthsFItalyHACNIT7/25/16E. cloacaeAMC-FEP-CTX-CAZ-GEN-IPM-TZP-MEMblaVIMUT
a Strains isolated from the same patient were indicated with the same letter (e.g., 137-F and 153-F); b mths (months); c CAR (Central African Republic), S. Leone (Sierra Leone); d HAC (hospital-acquired colonization), CAC (community-acquired colonization); e NIT (neonatal intensive unit), NSU (neonatal surgery unit), SNU (sub-intensive neonatal unit), CICU (cardiac intensive care unit); f amikacin (AMK), amoxicillin–clavulanic acid (AMC), cefepime (FEP), cefotaxime (CTX), ceftazidime (CAZ), ciprofloxacin (CIP), gentamicin (GEN), imipenem (IPM), meropenem (MEM), piperacillin–tazobactam (TZP), tigecycline (TGC), trimethoprim-sulfamethoxazole (SXT); g PBRT (profiles were sorted numerically from 1 to 49); UT = untypeable.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Agosta, M.; Bencardino, D.; Argentieri, M.; Pansani, L.; Sisto, A.; Ciofi Degli Atti, M.L.; D’Amore, C.; Putignani, L.; Bagolan, P.; Iacobelli, B.D.; et al. Prevalence and Molecular Typing of Carbapenemase-Producing Enterobacterales among Newborn Patients in Italy. Antibiotics 2022, 11, 431. https://doi.org/10.3390/antibiotics11040431

AMA Style

Agosta M, Bencardino D, Argentieri M, Pansani L, Sisto A, Ciofi Degli Atti ML, D’Amore C, Putignani L, Bagolan P, Iacobelli BD, et al. Prevalence and Molecular Typing of Carbapenemase-Producing Enterobacterales among Newborn Patients in Italy. Antibiotics. 2022; 11(4):431. https://doi.org/10.3390/antibiotics11040431

Chicago/Turabian Style

Agosta, Marilena, Daniela Bencardino, Marta Argentieri, Laura Pansani, Annamaria Sisto, Marta Luisa Ciofi Degli Atti, Carmen D’Amore, Lorenza Putignani, Pietro Bagolan, Barbara Daniela Iacobelli, and et al. 2022. "Prevalence and Molecular Typing of Carbapenemase-Producing Enterobacterales among Newborn Patients in Italy" Antibiotics 11, no. 4: 431. https://doi.org/10.3390/antibiotics11040431

APA Style

Agosta, M., Bencardino, D., Argentieri, M., Pansani, L., Sisto, A., Ciofi Degli Atti, M. L., D’Amore, C., Putignani, L., Bagolan, P., Iacobelli, B. D., Dotta, A., Martini, L., Di Chiara, L., Magnani, M., Perno, C. F., Andreoni, F., & Bernaschi, P. (2022). Prevalence and Molecular Typing of Carbapenemase-Producing Enterobacterales among Newborn Patients in Italy. Antibiotics, 11(4), 431. https://doi.org/10.3390/antibiotics11040431

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop