https://orcid.org/0000-0002-7070-8261
Figures
Abstract
Background
Klebsiella spp. can colonize the intestine of preterm neonates, and over-growth has been associated with necrotizing enterocolitis, hospital-acquired infections, and late-onset sepsis. This could lead us to suggest that the clinical pertinence of intestinal colonization with ESBL in preterm neonates appears to be important. We conducted this study to characterize the genetic proprieties of ESBL-producing Klebsiella pneumoniae (ESBL-KP) under clinical isolates and to describe the risk factors for the intestinal tract acquisition event during hospitalization.
Methods
One hundred and thirteen premature infants were recruited from the neonatal intensive care unit (NICU). All newborns are issued from the birth suites of the pregnancy department. Two rectal swabs were planned to define K. Pneumoniae intestinal carriage status. ESBL-KP was confirmed by Brilliance ESBL selective chromogenic Agar. Antimicrobial susceptibility testing including phenotypic testing and genotypic detection of the most commonly described ESBL genes was done. Logistic regression models were performed to find the variables associated with the acquisition event of ESBL-KP.
Results
A total of 62 (54.86%) premature neonates were colonized with ESBL-KP. The rate of blaSHV, blaTEM, blaCTX-M1, blaCTX-M2, blaCTX-M9, and blaOXA-48 genes among the isolates was 82, 48, 93.5, 4.8, 11.2 and 3.22%, respectively. We found that ESBLs K. Pneumoniae isolates were 100% resistant to amoxicillin, clavulanic acid-amoxicillin, cefotaxime, ceftazidime, and gentamicin. The regression model is for a given significant association between the tract intestinal of ESBL-KP acquisition events and the use of enteral tube feeding (OR = 38.46, 95% CI: 7.86–188.20, p-Value: 0.001), and endotracheal tubes (OR = 4.86, 95% CI: 1.37–17.19, p-Value 0.014).
Citation: Moussa B, Oumokhtar B, Arhoune B, Massik A, Elfakir S, Khalis M, et al. (2023) Gut acquisition of Extended-spectrum β-lactamases-producing Klebsiella pneumoniae in preterm neonates: Critical role of enteral feeding, and endotracheal tubes in the neonatal intensive care unit (NICU). PLoS ONE 18(11): e0293949. https://doi.org/10.1371/journal.pone.0293949
Editor: Monica Cartelle Gestal, LSU Health Shreveport, UNITED STATES
Received: September 15, 2022; Accepted: October 5, 2023; Published: November 7, 2023
Copyright: © 2023 Moussa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting information files.
Funding: The authors received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Klebsiella Pneumoniae healthcare-associated infections are a serious problem for neonate infants hospitalized in neonatal intensive care units NICU [1]. This pathogen arouses the interest of infection control specialists due to the increasing number of antibiotic-resistant strains. Indeed, more than a third of Klebsiella. pneumoniae isolates reported to the European Centers for Disease Prevention and Control were resistant to at least one antimicrobial group, The aminoglycosides are the most common resistance profile with combined resistance to fluoroquinolones and third-generation cephalosporins [2]. Antibiotic-resistance genes can be exchanged between gram-negative bacteria through horizontal gene transfer. The production of other types of ESBL, CTX-M like, more selective to cefotaxime than to other broad-spectrum cephalosporins, has been increasingly detected in the last years and is also increasing in K. pneumoniae [3–5].
Due to the combination of the Newborn’s immature immune system’s decreased functioning and frequent antibiotic exposure, hospitalized neonates are particularly susceptible to developing k.pneumoniea infections [6]. Moreover, it´s well known that the appearance of late infections in preterm infants is strongly associated with intestinal carriage, and colonized patients act as a reservoir for the spread of resistant organisms [7].
The first Bacterial colonization of the neonatal gastrointestinal tract occurs from the mother and the immediate inanimate environment [8,9]. However, some early clinical factors that might disrupt the normal acquisition process of the first microbiota in the neonate during NICU stay have been identified [10,11], such as prematurity (gestational age <37 weeks), length of stay, previous use of antibiotics, absence of breastfeeding, and Nasogastric tube feeding [12–14]. However, in contexts where the resistant bacteria have attained endemic levels, the dynamics of the spread seem more dependent on the environmental factors in the NICUs. This might lead us to suggest that the clinical pertinence of ESBL gut colonization of preterm neonates appears to be important, between 12 and 50% of newborns colonized with ESBL-producing bacteria have developed a bloodstream infection with positive blood cultures [15,16].
This study aimed to determine the intestinal tract acquisition of ESBL-KP prevalence in preterm neonates, describe their genetic properties, and study the main risk factors associated with colonization events during neonatal intensive care.
Materials and methods
Study design
This prospective study was conducted at the neonatology and intensive care unit (NICU) department of the University Hospital of Fez (Morocco) during 1 year from February 2019 to July 2020. The setting was a medical and surgical neonatal intensive care unit with 18 beds divided into 2 sectors (9 beds in each); the first sector corresponds to an intensive care unit and the second to a unit for preterm babies. This NICU is the only one in Fez city (the center of Morocco), with approximately 1.5 million inhabitants. Three seniors, 8 physicians, and 6 nurses are assigned to this ward daily.
According to the WHO criteria, we selected all premature infants hospitalized in the NICU to be studied for intestinal carriage, and prematurity was defined as any baby born alive before the end of 37 weeks of pregnancy [17]. Therefore, neonates hospitalized during the weekend who have died or output before 48h of hospitalization were excluded. Preterm infants were screened (a) at admission and (b) during hospitalization for ESBL-KP intestinal carriage. Only preterm infants who were not positive for a given species on admission were considered at risk of acquiring ESBL-KP.
Ethical consideration
The study protocol was approved by the Ethics Committee of the Faculty of Medicine and Pharmacy, and the HASSAN II University Hospital of Fez of Morocco, all the parents’ babies were informed of the conditions related to the study and gave their written, informed consent. Parents may remove their consent at any time during the study.
Data collection
Information was obtained from the medical information system and classified as follows: Anamnestic data (gestational age, delivery mode, prolonged premature rupture of membrane history (PPROM), postpartum clinical data (gender, birth weight, associated pathology, prematurity), and possibly evolving clinical data (invasive procedures, length of stay, antibiotic exposure, endotracheal tube and type of feeding).
Sampling and screening
Two rectal swabs were collected from each preterm newborn. The initial sample was performed up to 6 hours from admission to the NICUs and the second one after 5 days of hospitalization. Rectal swab specimens were enriched in nutrient broth BHI (Brain Heart infusion, Oxoid1) at 37°C for 24h. Then, they were inoculated on Mac Conkey agar plates and then incubated at 37°C for 24h. The identification of Enterobacteriaceae isolates was performed by classical bacteriological techniques.
Antimicrobial susceptibility testing
The antibiotic resistance patterns of isolates of K. pneumoniae were determined by disc diffusion method in agar according to the EUCAST for 14 discs of antibiotics including amoxicillin (10 μg); amoxicillin/clavulanic acid (20/10 μg); cephalothin (30μg); cefotaxime (30 μg); ceftazidime (30 μg); ertapenem (10 μg); nalidixic acid (30 μg); ciprofloxacin (5 μg); norfloxacin (10 μg); gentamicin (10 μg); amikacin (30 μg); Fosfomycin (50 μg); and cotrimoxazole (1.25/ 23.75 μg) used the disk diffusion on Mueller–Hinton agar (Bio-Rad, Hercules, CA). ESBL-KP was confirmed by a selective chromogenic medium for the screening of Extended Spectrum-Lactamase-producing Enterobacteriaceae (Brilliance ESBL Agar, Oxoid). The K. Pneumoniae strain ATCC 700603 was used as a quality control antibiogram control strain for ESBL production.
Molecular analysis
The DNA extraction was performed according to the method described in the previous studies [18]. In addition, an aliquot for 2μL of the supernatant was used as a DNA template for the PCR.
All ESBL-KP strains were screened by polymerase chain reaction (PCR) for the following β-lactamase-encoding genes: blaCTX-M, phylogenetic lineage groups 1, 2, and 9; blaTEM; blaSHV; blaKPC, blaNDM, blaVIM and blaOXA-48 "Table 1". Amplification reactions were carried out in a 50 μl volume containing 2 μl of DNA template, 2.5 mM MgCl2, 0.4 mM of each forward and reverse primer, 100 mM of each dNTP, and 2 U of Taq DNA Polymerase (Promega, Madison, WI) in PCR buffer performed and provided by the manufacturer. The known β-lactamase-producing strains E. coli U2A1790 (CTX-M-1), E. coli U2A1799 (CTX-M-9), Salmonella sp. U2A2145 (CTX-M-2), Salmonella sp. U2A1446 (TEM-1 and SHV-12) were used as positive controls. PCR products were detected on 1.5% agarose gels (FMC BioProducts, Rockland, ME) stained with ethidium bromide and visualized under Ultra Violet light.
DNA Sequencing
All amplified products obtained were sequenced to confirm their identification. Both strands of the purified amplicons were sequenced on a 3130 1 Genetic Analyzer (Applied Biosystems, Foster City, CA) using the identical primers that were used for PCR amplification. The nucleotide and protein sequences were analyzed using software from the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov).
Statistical analysis
Potential risk factors associated with ESBL-KP colonization were studied. Statistical analysis was performed with SPSS version 26, where additional variables were created for the analysis. Frequencies (percentages) of qualitative variables and mean values (standard deviation) of continuous were calculated. The chi-square test for continuous variables and Fisher’s exact test for categorical and nominal variables were used to make variable comparisons for cases with versus without colonization. Logistic regression models were used to identify variables related to ESBL-KP acquisition events. Multivariate adjusted odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were estimated and adjusted for: Admission age (day), gender (male, female), birth Weight (g) (< 2500 g, ≥ 2500 g), delivery mode (vaginal birth, cesarean section), invasive procedure (endotracheal tube, enteral tube feeding), antimicrobial exposure 3rd CG, feeding (breast milk, milk formula) and PPROM History (no PPROM, PPROM> 18 hours). All analyses were 2-tailed, and a p-level of < 0.05 was considered statistically significant.
Results
Population characteristics
One hundred and thirteen preterm infants were prospectively recruited for one year. Male neonates made up the majority of their 66 study participants (58.40%). The majority of 98 (86.72%) premature newborns had a birth weight of less than 2500 g and a median of 1736.12 g (IQR: 1400–2150). 83 (73.45%) of the newborns were delivered by cesarean section. 62 (54.86%) were born very preterm (gestational age: 28 to <32 weeks) and 87 (76.99%) with a respiratory distress complication “Table 2”.
Microbiological results
Of the 113 preterm newborn infants included in the study, 62 (54.8%) premature infants acquired ESBL-KP. The molecular and antibiotic susceptibility data of ESBL-producing K. Pneumoniae isolates are shown in “Table 3”. We found that ESBLs K. Pneumoniae isolates were 100% resistant to amoxicillin, clavulanic acid-amoxicillin, cefotaxime, ceftazidime, and gentamicin. The most significant resistance was observed in the TEM β-lactamases category to nalidixic acid, norfloxacin, and/or ciprofloxacin, sulfamethoxazole with (84.6%) followed by the CTX-M-2 β-lactamases category (60%). The resistance pattern of the SHV β-lactamases K. Pneumoniae isolates to nalidixic acid, norfloxacin, and/or ciprofloxacin, sulfamethoxazole, ertapenem was 46.1%,43.5%,46.1%, and 2.5%, respectively and two strains characterized as OXA-48 showed resistance to ertapenem. However, the data indicated that all ESBL isolates from K. Pneumoniae showed susceptibility to amikacin.
Risk factors analyses
The bivariate analysis suggested that type of delivery, previous antibiotic use, Invasive procedures, and feeding were significantly associated with the acquisition event, P < 0.05; “Table 2” ». Out of the 83 neonates with Caesarean delivery mode who have been admitted to the neonatal intensive care unit (NICU), 50(80.64%) acquired ESBL-KP compared to 12(19.35%) of those with Vaginal delivery mode (P = 0.045). It was also noted that neonates born to mothers without PPROM history were more likely to be colonized with ESBL-KP than those born to mothers with a PPROM history of more than 18 hours 51(82.25%) vs. 11(17.74%), P = 0.391. For the invasive procedure, the preterm neonates with an endotracheal tube and an enteral tube feeding probe have a high ESBLs K. pneumoniae colonization rate of 45 (72.58%)–60 (96.77%), P = 0.001, respectively. Preterm neonates exposed to third-generation cephalosporin antibiotics have acquired ESBL-KP at a lower rate than those who did not, 38 (65.51%) vs. 58(51.32%), P = 0.023 “Table 2”. All the variables analyzed in the bivariate analysis were associated with the risk of colonization (P < 0.25) and were therefore examined in the multiple logistic regression analysis.
The multivariate regression analysis reveals that premature infants with enteral tube feeding and endotracheal tubes have a significantly higher risk of acquiring ESBL-KP (p value = 0.001). Premature infants with caesarean section mode had a higher risk compared to premature with vaginal birth (OR = 2.27, 95% CI: 0.96–5.33, p-Value = 0.059). Among the antimicrobial risk factors, premature infants who were exposed to third-generation cephalosporins during hospitalization had a higher risk of acquiring ESBL-KP compared with preterm infants who were not exposed (OR = 2. 45, 95% CI: 1.14–5.24, p-Value = 0.020), and a moderate risk was observed in preterm infants who received milk formulae compared with those who received breast milk (OR = 0.28, 95% CI: 0.11–0.74, p-Value = 0.010). After adjustment for potential confounders, “Table 4”, premature neonates who received enteral tube feeding had a significantly higher risk of ESBL-KP acquisition than neonates who did not receive enteral tube feeding (OR = 38.46, 95% CI: 7.86–188.20, p-value < 0.001). Compared to neonates without endotracheal tubes, premature babies with endotracheal tubes had a significantly higher risk of ESBL-KP acquisition (OR = 4.86, 95% CI: 1.37–17.19, p-value <0.014).
There was no significant association in the multivariate analysis with ESBL-KP acquisition risk for all prematurity levels, Age at admission, gender, feeding, birth weight (< 2500 g, ≥ 2500 g), Delivery Mode, and antimicrobial exposure.
Discussion
K. pneumoniae infections are particularly troublesome among hospitalized newborns [23]. This strain is the most common cause of sepsis and epidemics in perinatal intensive care units [1,24]. Several reviews have highlighted that hospitalized patients carrying K. pneumoniae at the intestinal level developed fulminant infections with the same strain of carriage [25,26]. For one year, we investigated the molecular characterization of acquired K. Pneumoniae in the intestinal tract of hospitalized premature infants. Among 113 preterm infants included in this study, 62 (54.8%) acquired ESBL-KP. Premature infants are known to have an abnormal gut colonization pattern in the first few weeks of life [27,28], which may lead to an increased susceptibility to disease [29,30]. Compared to full-term infants, the gut microbiota of preterm infants has significantly reduced bacterial diversity and an abundance of microorganisms typically associated with hospital environments [31,32]. In this study the resistance profile genes result of ESBL-KP are presented below: SHV β-lactamases 51 (82%), TEM β-lactamases 30 (48%), CTX-M-1 β-lactamases 58 (93.5%), CTX-M-2 β-lactamases 03 (4.8%), CTX-M-9 β-lactamases 07(11.2%) and OXA-48 β-lactamases 02 (3.22%), This high degree of prevalence has been frequently reported in investigations focused on gut colonization [1,33]. Still, this molecular diversity is not surprising since opportunistic microorganisms K. pneumoniae are known to acquire resistance genes through antibiotic pressure [34].
To our knowledge, this is the first study in our country that identified endotracheal intubation and enteral tube feeding as independent risk factors for colonization of neonates with ESBL-KP both were significant in the multiple regression analysis (p-value = 0,001). Invasive procedures are usually associated with ESBL-KP colonization and/or infection among hospitalized neonates [35]. Other studies reported that the method of feeding, as well as the nature of enteral feeds, are important factors in early gut colonization [16,36]. Petersen et al. have shown that enteral tube feeding resulted in a high bacterial density on the first day of use [37]. Also, several studies show that the bacterial flora in the feeding tubes of newborns can influence the bacterial colonization of the intestinal tract [12,38].
However, Crivaro et al. [39] and Cassettari et al. [40] observed that empirical antibiotic treatment is a significant risk factor associated with ESBL-KP employment status in newborns. Our results confirmed this, in the univariate analysis and unadjusted regression, especially for the third generation of cephalosporins exposed under ESBL-KP acquisition. By performing a multi-logistic regression analysis, we found limited associations with early empirical antibiotic therapy. It seems that not antibacterial therapy itself but poor hospital hygiene enables the circulation and transmission of multiresistant strains, which then requires broad-spectrum antibacterial agents and results in longer NICU length of stay and greater potential for colonization by resistant organisms [41,42].
Previous studies have also emphasized the role of both anamnestic and infant-related risk factors, such as PPROM, vaginal birth, birth weight, preterm birth rate, and length of stay, for the acquisition of ESBL-KP [17–19]. These findings were not seen in the present study and could presumably be attributed to the small number of cases.
Moreover, it seems that ESBL Enteroacteriacae colonized mothers are an independent risk factor for the colonization of neonates with ESBL-Enterobacteria. She might harbor these bacteria in their normal intestinal flora and contaminate their newborns during birth. To decrease neonatal morbidity and mortality, several studies suggested systematic screening of the intestinal flora of premature newborns and their mothers, should be implemented in neonatal wards [2,19].
Limitations of our study include the small sample size; therefore, inference about causality is limited. in addition, it is unusual that a study could identify all the risk factors associated with the acquisition of these multi-resistant bacteria that play an essential role in causing infection in NICUs. The difficulty of the subject is due to the complexity of tracking newborns after birth. Many factors limit studies in this regard, including operating costs, deaths, short hospitalization times, and the difficulty of following anamnestic and biological clinical data [33,43].
Conclusion
The acquisition of ESBL-KP carriage was demonstrated in 54.8% of preterm infants. Our finding supposes that enteral tube feeding and endotracheal tubes may be independent risk factors for colonization in the intestinal tract with ESBL-KP during a hospital stay in the neonatal intensive care unit. This highlights the current status of two practices that will require procedural updating in the NICU.
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