Multidrug-resistant ESBL-producing Enterobacteriaceae and associated risk factors in community infants in Lebanon

Introduction: Extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-PE) infections are a growing threat to children, and the treatment of these infections becomes more and more challenging. A huge reservoir for ESBLs in the community is the fecal flora of children. This study investigates the rectal colonization, associated risk factors, antimicrobial susceptibility, and molecular characterization of ESBL-PE in Lebanese community infants. Methodology: A total of 117 rectal swabs were taken from healthy infants between 1 and 12 months of age. Detection of ESBLs was carried out using the double-disk synergy test, combination-disk method, and multiplex polymerase chain reaction (PCR). A questionnaire about the infant’s history and risk factors for carrying ESBL-PE was administered. Results: In total, 58 (49.6%) of 117 participants were ESBL-PE carriers. Some significant important risk factors for colonization in this study were male gender, hospital birth, caesarean delivery, and being formula-fed. Observed decrease in colonization rate was associated with intimate hygiene habits. Carriers of multiple bla genes were the most common. CTX-M type was the major harbored, gene and CTX-M-9 was the most predominant, followed by CTX-M-15 type. Conclusions: To the best of our knowledge, this is the first available data about the carriage rate of ESBL-PE in community infants in Lebanon and the Middle East, the first study showing that birth in hospital, caesarean delivery, and being formula-fed are all significantly associated risk factors for the high colonization rates in community – not hospitalized – infants, and showing the dominance of multiple resistance gene carriage and wide dissemination of CTX-M-9 ESBL.


Introduction
Extended-spectrum beta-lactamase (ESBL)producing bacteria is a major worldwide threat among drug-resistant bacteria in both hospital and community settings [1].ESBLs can hydrolyze all penicillins and cephalosporins, including the extended-spectrum cephalosporins such as cefotaxime or ceftazidime [2].Many ESBL producers are multi-resistant to non-betalactam antibiotics, including fluoroquinolones and aminoglycosides [3], trimethoprim, tetracyclines, sulfonamides, and chloramphenicol, and this is often encoded by the gene harbored by the same plasmids that determines the ESBL type [4].Effective antibiotic therapy for treating these infections is limited to a small number of drugs [5] such as carbapenems; as a result, the chance of resistance to carbapenems among the Enterobacteriaceae increases.There are more than 1,600 known beta-lactamases, a list that is rapidly expanding [6].TEM, SHV, and CTX-M-type of ESBLs are most often found in a wide range of Enterobacteriaceae, with increasing frequency [7][8][9].However, the majority of ESBL-producing strains are Escherichia coli and Klebsiella pneumoniae [10].TEM-and SHV-type beta-lactamases, mainly produced by K. pneumoniae, have spread throughout hospital settings, and CTX-M enzymes, mainly produced by E. coli, have become predominant in the community [9].CTX-M enzymes were identified in 1989 from E. coli isolates in Germany [11], but they did not become predominant over the other ESBL enzymes until the first decade of the twenty-first century, during which an extraordinary spread of these enzymes was observed [10] in both hospital and community settings [12].Plasmids encoding blaCTX-M-15 are found mainly in Enterobacteriaceae and were recently named plasmids of resistance responsible for outbreaks because of their capacity to acquire genes of resistance and to transfer among bacteria [13].Intestinal colonization by ESBLproducing isolates may thus represent a reservoir for ESBLs in the community not detected in clinical isolates [14].The clinical impact of ESBL-producing pathogens on morbidity and mortality in infectious diseases in adults, as well as their economic burden, are well documented [15,16].In young infants, Gramnegative organisms are the most common cause of serious bacterial infection [17,18].Options for treatment of these infections are generally limited, and given that fewer antibiotics are approved for use in children, the problem is critically important to address [19].
It is critical to better define the prevalence, risk factors, and molecular characterization of ESBLproducing organisms carried by infants to adopt bestpractice infection control measures and help in the appropriate choice of empirical antimicrobial coverage for infections in these populations.The aim of this study was to investigate the prevalence and predisposing factors of intestinal carriage of ESBL-producing Enterobacteriaceae (ESBL-PE) among healthy Lebanese community infants and to characterize the resistance genes blaTEM, blaCTX, blaSHV, blaCTX-M-2, blaCTX-M-9, and blaCTX-M-15.

Sample population and questionnaire
A total of 117 healthy infants between 1 and 12 months of age who were brought to clinics for vaccination were chosen from 3 different clinics in Lebanon for this study.A questionnaire was completed for each participant, which included name, age, gender, hospital or home birth, vaginal or cesarean delivery, premature or low birth weight, extended hospital stay after birth, breast-or formula-fed, previous antibiotic or antacid treatment, previous hospital admission, previous urinary tract infection, contact with pets, and intimate hygiene habits.This investigation was performed between January and May 2013.

Bacterial isolation and ESBL detection and confirmation
From each participant, a rectal swab was taken using a sterile swab moistened with sterile saline and was immediately plated on MacConkey agar plates (Oxoid, Hampshire, UK) supplemented with 2 mg/L ceftazidime (Oxoid, Hampshire, UK) within 5 days of preparation.Selective media showing growth colonies were selected for subsequent characterization.Bacterial identification was performed using Gram staining, biochemical testing (indole, methyl red, Voges-Proskauer, citrate, and urease), and the API 20E system (bioMerieux, Marcy l'Etoile, France).
The isolates were first screened for ESBL production using ceftazidime, cefepime, cefotaxime, cefpodoxime, ceftriaxone, and aztreonam disks (Oxoid, Basingstoke, UK).Next, phenotypic confirmatory tests were carried out by the double-disk synergy test and combination-disk method.The double-disc synergy test was performed on agar with a 30 µg disk of cefotaxime, ceftriaxone, ceftazidime, and a disk of amoxicillinclavulanate (containing 10 µg of clavulanate) positioned at a distance of 20 mm (center to center).The test was considered positive when a decreased susceptibility to cefotaxime was combined with a clearcut enhancement of the inhibition zone of cefotaxime in front of the clavulanate-containing disk.The combination-disk method was performed using ceftazidime, ceftazidime-clavulanic acid, cefotaxime, and cefotaxime-clavulanic acid.The organisms were considered to be ESBL producing when a ≥ 5 mm increase in a zone diameter for either antimicrobial agent tested in combination with clavulanic acid compared with the zone diameter of the agent when tested alone [20].Molecular analysis of all positive isolates screened was done.

Characterization of genes encoding ESBLs
Multiplex polymerase chain reaction (PCR) was performed to detect TEM, SHV, and CTX-M genes.Crude genomic DNA was extracted from the isolates by the heat lysis method.One pure colony was suspended in 40 µL of sterile distilled water, and the cells were lysed by heating at 95°C for 5 minutes, then centrifuged, and cooled to 4°C [21].Suitable primers [22] (Sigma-Aldrich, Taufkirchen, Germany), each targeting selected regions of the blaTEM, blaSHV, and blaCTX-M genes were used (Table 1).
Amplification reactions were performed in a final volume of 25 μL containing 12.5 μL PCR Master Mix 2X (Thermo Scientific, Lithunia, EU) and 12.5 μL of DNA, primers, and H2O.A 12.5 μL PCR master mix reaction buffer, and 0.5 μL TEM F, 0.5 μL TEM R, 1 μL of each remaining primers (10 μM/μL), 2.5 μL H2O, and 5 μL of the template DNA preparation were added to the reaction mixture.Reactions were performed in a DNA thermal cycler (BIOER, Tokyo, Japan) under the following conditions: denaturation at 94°C for 5 minutes followed by 30 cycles at 94°C for 20 seconds, 61°C for 30 seconds, and 72°C for 1 minute with a final extension of 72°C for 5 minutes [23].
Reactions were performed in a DNA thermal cycler under the following conditions: denaturation at 94°C for 5 minutes followed by 30 cycles at 94°C for 15 seconds, 56°C for 15 seconds and 72°C for 45 seconds with a final extension of 72°C for 5 minutes.
After PCR amplification, 2.5 μL of each reaction was separated by electrophoresis in 1.5% agarose gel for 30 minutes at 100 V in 0.5 × TBE buffer.DNA was stained with ethidium bromide (1 μg/mL), and the bands were detected using a UV transilluminator (Cleaver Scientific Ltd, Rugby, UK.).

Statistical analysis
The data were analyzed using Yates's corrected χ 2 test.P values < 0.05 were taken as significant.Statistical analysis was performed using Minitab software.

Factors associated with ESBL-PE carriage
The analysis of risk factors for fecal carriage in healthy infants who participated in this study is shown in Table 2.A significantly high carriage rate was found among infants who were born in hospitals (52.3%) compared with 0% for those who were born at home (p = 0.027).Regarding the type of delivery, caesarean delivery was observed to be associated with an increased risk of carriage, and this was statistically significant (p = 0.036).Formula-fed infants were found to be significantly associated with a higher ESBL-PE carriage rate (60.5%) than those who were breast-fed (p = 0.034).Other associated risk factors were extended hospital stay after birth, low birth weight, previous antibiotic or antacid intake, previous hospital admission, and previous urinary tract infection, although the differences were statistically insignificant.Premature birth and contact with pets were not observed to be associated with ESBL-PE carriage in this study.With respect to intimate hygiene habits, infants whose parents used water or soap and water, and then dried with tissue after each diaper change showed a reduced ESBL-PE carriage rate (43.7%), compared to 56.6% for those whose parents used only dry tissue or wipes (p = 0.08) (Table 2).

Discussion
Community-acquired ESBLs in healthy children accounted for 22% of infections in the United States [24].The association between fecal carriage in children and risk for subsequent infection with an ESBL Enterobacteriaceae has been reported [25].To the best of our knowledge, this survey was the first conducted in Lebanon and the Middle East concerning the intestinal carriage of ESBL-PE in the healthy infant community.The prevalence was found to be 49.6% in this age group (1-12 months), which is a high rate compared to other studies among community and hospitalized pediatric patients from different countries and to a previous study in 2005 in an adult population in Lebanon, where the ESBL-PE carriage rate was 2.4% [26].Intestinal carriage by ESBL-producing Gram-negative bacteria in healthy French and Swedish children ranged from 2.9% to 6.7%, with CTX-M-15, CTX-M-14, and CTX-M-1 constituting the majority of ESBLs [27,28].Twentyfour percent (30/125) of healthy Spanish children were colonized by ESBL-producing strains, and CTX-M-1 was the most common type [29].Rare reports are available about carriage in healthy children in Asia, Africa, and the Middle East, and there are very few investigations about carriage in hospitalized children [30].ESBL-PE strains were detected in 12% (6/50) of fecal samples collected from the inpatients of a Japanese pediatric hospital, and all the ESBLs belonged to the CTX-M-1 group [31].The carriage rate of ESBLproducing E. coli strains isolated from children warded in a Malaysian tertiary hospital was 19.1% (21/110), and CTX-M-15 was the predominant type [32].ESBLs were identified in 13.4% (18/143) of E. coli isolates from Libyan children's stools, and all isolates that produced ESBLs belonged to CTX-M type [33].Among children living in a very remote Senegalese village, 10% (2/20) were found to be fecal carriers of a multi-resistant E. coli clone that produced CTX-M-15 [34].In a tertiary care center in Turkey, fecal carriage of ESBL-producing E. coli and Klebsiella spp. was 24% (66/270) in hospitalized pediatric patients and 7.2% (14/194) in ambulatory children [35].Our investigation showed that the majority of ESBL-PE isolated from healthy Lebanese infants carried multiple bla genes and that blaCTX-M was the predominant (91.4%) gene.TEM type was also considered a frequent (67.2%) disseminated beta-lactamase type, as the majority of the CTX-M-producing isolates (69.8%) were found to be co-producers of the TEM and SHV genes; either two or all three genes occurred together.Although epidemiological studies report that CTX-M-type ESBLs are endemic in most European, Asian, and South American countries, with high rates of prevalence ranging from 30% to 90% for E. coli [36], TEM enzymes could also spread in community settings.
The mechanisms of CTX-M emergence are more complex than those of TEM-or SHV-type ESBLs, involving not only a clonal spread of bacteria but also the spreading of enzymes encoded by genes harbored by plasmids and/or other mobile genetic elements, which promotes their epidemiological success.Furthermore, all strains of CTX-M-producing Enterobacteriaceae are also (most of the time) resistant to other families of antibiotics, especially aminoglycosides and fluoroquinolones, as plasmidencoded CTX-M often carry other genes of resistance (particularly to aminoglycosides, tetracyclins, sulfamides, and trimethoprim), suggesting coresistance, co-expression, and co-selection, which makes carbapenem use mandatory [37].
CTX-M-15 is the most frequently found CTX-Mtype ESBL in E. coli worldwide, involved in community-acquired infections as well in nosocomial infections [38], and has been responsible for various outbreaks, especially in France, Great Britain, Canada, Spain, and Tunisia [38,39].In the present study, molecular characterization of CTX-M-type ESBLpositive isolates revealed that a greater number of isolates (54.7%) co-harbored either two or three CTX-M-type genes (blaCTX-M-9, blaCTX-M-15, and blaCTX-M-2).CTX-M-9-type was the predominant (86.8%) type, followed by CTX-M-15 (49.1%).Some CTX-M is specific to some countries (e.g., CTX-M-9 and CTX-M-14 in Spain, CTX-M-1 in Italy, or CTX-M-2 in South America and Japan), while CTX-M-15 is distributed worldwide [12].These data indicate that the epidemiology of colonization with ESBL-PE in the community is complex, and the source of acquisition and transmission of these genes may be from both hospital and community settings; further complex studies are needed.
All ESBL-PE isolated from Lebanese community infants were susceptible (100%) to imipenem, and 93.1% were susceptible to amikacin; this is considered positive regarding absence of metallo-beta-lactamases among these isolates.However, most isolates were resistant to other antimicrobials tested.High resistance to nalidixic acid (63.8%), tetracycline (60.3%), gentamicin (48.3%), and trimethoprimsulfamethoxazole (55.2%), was found; this result confirmed the carriage of multidrug-resistant ESBL-PE in asymptomatic healthy infants in Lebanon.This high carriage rate of ESBL-PE and associated resistance to aminoglycosides and trimethoprim-sulfamethoxazole, as well as high frequency of co-existence of fluoroquinolone, increases the risk of infection with multidrug-resistant bacteria, which results in the need for last-resort antibiotics, such as carbapenems and colistin, in the treatment of common infections [5].
Males appeared to have higher colonization rates than did females.This observation is difficult to explain and requires further exploration.
There have been reports on the risk factors for infection or colonization by ESBL-producing bacteria in children from pediatric intensive care units and neonatal intensive care units; the risk factors identified include artificial nails of hospital staff, cockroach infestation as vectors [40,41], younger gestational age, low birth weight, prolonged mechanical ventilation, longer hospital stay, invasive devices, antibiotic use [42][43][44][45], and mother-to-child transmission [46].
We investigated the risk factor for fecal carriage among participating infants and found that birth in a hospital and caesarean delivery were significantly associated with an increased risk of ESBL-PE carriage, suggesting that hospital environment or staff may be a source of these isolates.Infants with low birth weight, extended hospital stay after birth, previous antibiotic or antacid intake, previous hospital admission, and previous urinary tract infection were observed to have higher colonization rates, although this was statistically insignificant.On the other hand, we found that infants who were formula-fed had a significantly higher carriage rate (60.4%) than those who were breast-fed (43.2%), and this was statistically significant.This suggested that acquisition of ESBL-PE in this age group may be related to the milk source, water used, or to the practices around of feeding (e.g., hygiene, sterilization of bottles, etc.).Another important risk factor was observed in this investigation was intimate hygiene habits; infants whose intimate areas were cleaned with dry tissue or wipes after each diaper change had higher ESBL-PE colonization rates than their counterparts whose intimate area were washed with water or soap and water.Although this finding was not significant (p = 0.08), it could be further studied and explained.

Conclusions
This study sheds light on the ESBL-PE carriage rate in Lebanese community infants and establishes a basis for future surveillance of carriage.A relatively high overall rate compared to some other countries was found.The majority of isolated ESBL-PE harbored multiple genes (blaTEM, blaSHV, and blaCTX-M), where CTX-M was the predominant and CTX-M-9 and CTX-M-15 were the most widespread ESBL types.Significant risk factors for this high carriage were hospital birth, caesarean delivery, and being formulafed; these findings were observed for the first time.Hygiene measures and protocols are recommended in Lebanese hospitals to reduce transmission of ESBL-PE from staff, equipment, and environment to newborn babies; also, breastfeeding is recommended, as it decreases colonization rates.Further investigations are needed to evaluate the correlation between baby milk formula and ESBL-PE colonization, such as screening infant milk formula in the Lebanese market and screening water used in the preparation of these formulas for ESBL-PE.In addition, the relationship between intimate hygiene habits and decreased level of ESBL-PE carriage rate warrants further investigation.Alarming multidrug-resistant ESBL-PE is of great concern, especially in this age group, where treatment options are limited.Rational use of antibiotics, especially in pediatrics, is required.

Table 2 .
Analysis of the risk factors for extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-PE) fecal colonization in healthy Lebanese infants.

Table 3 .
Molecular characterization of 53 isolates harboring CTX-M ESBL type in community infants.