Fecal Carriage of Extended-Spectrum β-Lactamase-Producing Enterobacteriaceae Strains Is Associated with Worse Outcome in Patients Hospitalized in the Pediatric Oncology Unit of Beni-Messous Hospital in Algiers,Algeria

Abstract

Objectives:

The current study aimed to investigate extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E) fecal carriage in children with different cancers admitted in the pediatric oncology unit of Beni-Messous Hospital (Algiers, Algeria).

Materials and Methods:

Rectal swabs from children with cancer were sampled from February 2012 to May 2013 within 48 hours following their admission. After species identification and detection of ESBL production by double-disk synergy test (DD test), antibiotic susceptibility was determined by the standard disk diffusion method. Antibiotic resistance genes, including bla genes and plasmid-mediated quinolone resistance (PMQR) genes, were investigated by polymerase chain reaction (PCR). The phylogenetic grouping of Escherichia coli strains was determined by PCR.

Results:

Of the 171 children studied, 93 (54%) were ESBL carriers. An antibiotic treatment for the last 3 months before admission (p = 0.01), hematological malignancies (p = 0.003), and death (p = 0.0003) were more frequent in the ESBL-E group than in the non-ESBL group. Multivariate analysis showed that hematological malignancies (odds ratio [OR]: 3.9; confidence interval [CI]: 1.1–14.1; p = 0.04) and ESBL-E carriage (OR: 6.2; CI: 1.7–22.00; p = 0.005) were two independent factors associated with increased risk of death. A total of 103 ESBL-E isolates were obtained. Klebsiella pneumoniae and E. coli isolates were the most frequently isolated. PCR amplification showed that all the isolates produced a CTX-M ESBL (CTX-M-15, CTX-M-14, and CTX-M-3). The PMQR genes detected were qnrB, qnrS, and aac(6′)-Ib-cr. E. coli isolates were assigned to four major extraintestinal pathogenic E. coli phylogroups, including B2 and D.

Conclusion:

This study provides, for the first time, insight into epidemiology of the ESBL-E fecal carriage among children with cancer in Algeria.

Introduction

Antimicrobial resistance is of particular concern in cancer patients especially in relation to infection therapy and prevention.¹ The wide spread of extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E) is of special importance since it raises doubts about the efficacy of some noncarbapenem β-lactams as first-line treatment in cancer patients.^2,3

Recently, a significant increase in the prevalence of ESBL-E fecal carriage has been reported in a variety of settings, including community,⁴ hospitalized patients,⁵ newborns, infants,⁶ and healthy children,⁷ as well as in pediatric emergency.⁸

Intestinal colonization with ESBL-producing bacteria may serve as a reservoir of resistance genes that subsequently may be acquired by commensal strains of the microbial flora such as Escherichia coli and Klebsiella pneumoniae that can cause infections.⁸

In fact, ESBL-producing E. coli (ESBL-EC) fecal carriage is considered as a risk factor in bloodstream infections (BSI) with higher mortality compared with non-ESBL-EC in immunosuppressed patients.³ It has been shown that ESBL-E were more frequently isolated in the hematological malignancy group, as well the incidence of BSI was almost eight times higher in patients with hematological malignancies than in patients with solid tumors.^9,10

Even though ESBL-E fecal carriage has been largely investigated in adult patients with cancer,^3,10–12 little is known regarding fecal carriage of ESBL-E in pediatric oncology.^13,14 The current study aimed to investigate ESBL-E fecal carriage in children with different cancers admitted to the pediatric oncology ward of Beni-Messous Hospital (Algiers, Algeria).

Materials and Methods

Design of the study

This study was performed from February 2012 to May 2013 in the pediatric oncology unit of Beni-Messous Hospital. The unit had a capacity of 12 rooms and 36 beds. The study population comprised 171 children with different cancers; a questionnaire was completed for each participant regarding age, sex, previous admission, recent use of antibiotics, and type of cancer.

Definitions

Previous hospital admission was considered as a factor if hospitalization occurred within 3 months before the current stay. Recent antibiotic treatment was defined as an antibiotic treatment within 3 months before sample collection. Concomitant bacteremia due to ESBL-E was noted at the time of sample collection. The 30-day mortality rate was calculated by including all deaths occurring for any reason within 30 days of the hospital admission.

Microbiological analysis

Rectal swabs were taken within the first 48 hours of patient admission after receiving parental consent. Each swab was plated onto MacConkey agar supplemented with 4 μg/ml of either cefotaxime or ceftazidime and incubated at 37°C for 24 hours. After incubation, each morphologically different colony with Enterobacteriaceae appearance was selected for identification by matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI-TOF MS; IVD MALDI Biotyper version 2.3; Bruker Biospin SAS, Wissembourg, France).

Then, the isolates were screened for ESBL production using a double-disk synergy test (DD test).¹⁵ Isolates showing positive results with DD test were further tested by polymerase chain reaction (PCR) screening for ESBL determinants, as described below.

Antimicrobial susceptibility testing

Antibiotic susceptibility was determined on Mueller–Hinton agar using the standard disk diffusion procedure as described by the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST 2016).¹⁵ The following antibiotics were tested: piperacillin/tazobactam (30/6 μg), cefotaxime (5 μg), ceftazidime (10 μg), cefoxitin (30 μg), cefepime (30 μg), aztreonam (30 μg), imipenem (10 μg), ertapenem (10 μg), amikacin (30 μg), gentamicin (10 μg), tobramycin (10 μg), ciprofloxacin (5 μg), and cotrimoxazole (1.25/23.75 μg). Susceptibility patterns were interpreted according to the guidelines of EUCAST, Clinical breakpoints v.6.0. 2016.¹⁵

Phenotypic detection of carbapenemase

The modified Hodge test (MHT) was performed on a Mueller–Hinton agar plate with and without cloxacillin (250 μg/ml) for detection of carbapenemase activity, as previously described.¹⁶

Detection of antibiotic resistance genes

The bla_TEM, bla_SHV, and bla_CTX-M genes were screened by PCR and sequencing, as previously described.¹⁷ Carbapenemase-encoding genes bla_KPC, bla_VIM, bla_IMP, bla_NDM, bla_OXA-23-like, bla_OXA-24-like, bla_OXA-58-like, and bla_OXA-48-like were screened using a multiplex PCR, as previously described.¹⁸ The qnr, qepA, and oqxAB genes were screened by real-time PCR, as previously described.^19,20 The aac(6′)-Ib-cr determinant was detected by pyrosequencing.²¹

Phylogenetic groups and virulence genotyping of E. coli

PCRs were performed to determine the phylogenetic groups (A, B1, B2, C, D, E, F, and clade I) of E. coli isolates using the revised Clermont method.²²

Statistical methods

Univariate analysis of risk factors was performed using chi-square test and Fischer's exact test for qualitative variables and Student's t-test for quantitative variables. p-Values <0.05 were considered significant. For multivariate analysis, logistic regression analysis was used. Variables with a p-value <0.10 on univariable analysis were entered into the model procedure using a stepwise backward process. A p-value <0.05 was considered statistically significant.

Results

Characteristics of the studied population

During the study, 171 children with various types of cancers admitted into the pediatric oncology unit were screened for fecal ESBL-E carriage. The sex ratio (male/female) was 1.2 (93/78) and the mean age ± standard deviation was 5.2 ± 4.2 years.

Among the 171 children, 106 (62%) had hematological malignancies (87 acute leukemia and 19 lymphoma), 37 (22%) neuroblastoma, 16 (9%) nephroblastoma, and 12 (7%) different sarcoma. Of the 171 patients, 132 (77%) have been previously hospitalized and 125 (73%) patients had received antimicrobial therapy in the previous 3 months, especially cefotaxime alone or in combination with other antibiotics 104/125 (83%).

Factors associated with ESBL-producing Enterobacteriaceae fecal carriage

Of the 171 children with cancer enrolled in this study, 93 (54%) were ESBL-E carrier. Five patients had bacteremia due to an ESBL-E strain (5%, 5/93).

The ESBL-E colonized group was similar to the noncolonized group for age, gender, previous hospital admission, and bacteremia with ESBL-E (Table 1).

Table 1.

Comparative Characteristics of the Study Population According to the Extended-Spectrum β-Lactamase-Producing Enterobacteriaceae Fecal Carriage

	ESBL⁻ N (%), N = 78	ESBL⁺ N (%), N = 93	p
Age (years)
Mean ± SD	5.6 ± 4	4.7 ± 4.2	0.2
Range	1 month to 16 years	1 month to 15 years
Gender
Male	37 (47)	56 (60)	0.09
Female	41 (53)	37 (40)
Previous hospital admission	57 (73)	75 (81)	0.2
Antibiotic treatment for the last 3 months	50 (64)	75 (81)	0.01
Underlying disease
Hematological malignancies	38 (49)	68 (73)
Nephroblastoma	12 (15)	4 (4)
Neuroblastoma	23 (30)	14 (15)	0.003
Different sarcoma	5 (6)	7 (8)
Bacteremia with ESBL-E	2 (3)	5 (5)	0.3
Death	3 (4)	22 (24)	0.0003

ESBL-E, extended-spectrum β-lactamase-producing Enterobacteriaceae; SD, standard deviation.

An antibiotic treatment for the last 3 months before admission (75/93, 81% vs. 50/78, 64%, p = 0.01), hematological malignancies (68/93, 73% vs. 38/78, 49% p = 0.003), and death (22/93, 24% vs. 3/78, 4%, p = 0.0003) were more frequent in the ESBL-E group than in the non-ESBL group.

Among children with hematological malignancies (106), death was more frequent in ESBL-E carriers than in ESBL-E noncarriers (20/68, 29% vs. 2/38, 5%; p = 0.003). In binary logistic regression, the independent factors associated with increased risk of ESBL-E carriage were hematological malignancies (odds ratio [OR]: 2.8; confidence interval [CI]: 1.4–5.3; p = 0.002) and an antibiotic treatment for the last 3 months before admission (OR: 2.2; CI: 1.1–4.5; p = 0.03), and independent factors associated with increased risk of death were hematological malignancies (OR: 3.9; CI: 1.1–14.1; p = 0.04) and ESBL-E carriage (OR: 6.2; CI: 1.7–22.00; p = 0.005).

Bacterial isolates and antimicrobial susceptibility data

From 93/171 patients, 103 ESBL-E were isolated, including 49 K. pneumoniae, 38 E. coli, 12 Enterobacter cloacae, 3 Klebsiella oxytioca, and 1 Citrobacter braakii.

Table 2 shows the antimicrobial susceptibility pattern of 103 ESBL-E isolates. They were all susceptible to ertapenem and imipenem, except one isolate of E. cloacae was resistant to ertapenem. However, no carbapenemase production was observed in this isolate when tested by Hodge test on a Mueller–Hinton agar supplemented or not with cloxacillin.

Table 2.

Antimicrobial Susceptibility Pattern of Extended-Spectrum β-Lactamase-Producing (n = 103) Enterobacteriaceae Strains Isolated from Hospitalized Children Fecal Carriage

	Escherichia coli (n = 38), %	Klebsiella pneumoniae (n = 49), %	Enterobacter cloacae (n = 12), %	Klebsiella oxytoca (n = 3), %	Citrobacter braakii (n = 1), %
CTX	0	0	0	0	0
CAZ	16	6	8	33	0
FEP	8	20	0	0	0
ATM	8	6	0	0	0
FOX	97	100	NR	100	NR
TZP	92	67	75	67	100
IMP	100	100	92	100	100
ETP	100	100	92	100	100
TOB	29	26	8	0	0
GMI	29	26	33	0	0
AMK	74	61	58	33	100
CIP	45	73	50	100	100
SXT	13	10	0	0	0

AMK, amikacin; ATM, aztreonam; CAZ, ceftazidime; CIP, ciprofloxacin; CTX, cefotaxime; ETP, ertapenem; FEP, cefepime; FOX, cefoxitin; GMI, gentamicin; IMP, imipenem; NR, not reported; SXT, cotrimoxazole; TOB, tobramycin; TZP, piperacillin/tazobactam.

Characterization of β-lactamases and plasmid-mediated quinolone resistance genes

All strains were confirmed as ESBL producers by PCR amplification. The ESBL genes identified were bla_CTX-M-15 (n = 91/103; 88%), bla_CTX-M-14 (n = 6/103; 6%), and bla_CTX-M-3 (n = 6/103; 6%) (Table 3). No carbapenemase-encoding genes were detected by the multiplex PCR.

Table 3.

Prevalence of Extended-Spectrum β-Lactamases and Plasmid-Mediated Quinolone Resistance Genes in Enterobacteriaceae Isolates

	K. pneumonia, n = 49, (%)	E. coli, n = 38, (%)	E. cloacae, n = 12 (%)	K. oxytoca, n = 3 (%)	C. braakii, n = 1 (%)
ESBL genes
bla_CTX-M-15, n = 91	46 (94)	32 (84)	10 (83)	2 (67)	1 (100)
bla_CTX-M-14, n = 6	1 (2)	5 (13)	0 (0)	0 (0)	0 (0)
bla_CTX-M-3, n = 6	2 (4)	1 (3)	2 (17)	1 (33)	0 (0)
PMQR genes
qnrB, n = 26	18 (37)	0 (0)	7 (58)	0 (0)	1 (100)
qnrS, n = 1	1 (2)	0 (0)	0 (0)	0 (0)	0 (0)
aac(6′)-Ib-cr, n = 34	15 (31)	11 (29)	7 (58)	0 (0)	1 (100)

PMQR, plasmid-mediated quinolone resistance.

The qnr genes were detected in 27 isolates (26%), including qnrB (n = 26) and qnrS (n = 1) and the aac(6′)-Ib-cr gene in 34 isolates (33%) (Table 3).

Phylogenetic groups of E. coli isolates

ESBL-producing E. coli strains were mainly assigned to four major phylogenetic groups: D (15 isolates), A (7 isolates), B1 (8 isolates), and B2 (7 isolates). Only one isolate belonged to the phylogenetic group F. The B2 isolates produced CTX-M-15 (n = 6) and CTX-M-14 (n = 1). The D isolates produced CTX-M-15 (n = 11), CTX-M-14 (n = 3), and CTX-M-3 (n = 1).

Discussion

Colonization with ESBL-E, most commonly of the gastrointestinal tract (GIT), has been linked to subsequent infection with these resistant pathogens. This is particularly concerning in the setting of cancer as cytotoxic chemotherapy and extensive uses of broad-spectrum antibiotics alter the gut microbiome. The GIT can serve as a significant source of infecting organisms.^3,11,23 The ESBL-E prevalence increased dramatically worldwide over the last decade with variation between countries. In Algeria, no data are available about ESBL fecal carriage in children with cancer, so this is the first study demonstrating the alarming high prevalence of colonization by ESBL-E.

In our study, the prevalence of ESBL-E fecal carriage was (54%) similar to that reported by Thacker et al. in an oncology pediatric unit in India (58%),¹⁴ but higher than that reported (19%) in a pediatric oncology unit in the Czech Republic.¹³ It was also higher than those of other categories of children: 21% in children without cancer admitted in a pediatric unit in Madagascar,²⁴ 31% in children with severe acute malnutrition in Niger,²⁵ and 25% in healthy children in Lebanon.⁷

In the current study, the majority of ESBL-E was isolated from children with hematological malignancies (73%) compared with the children with solid tumors. The same fact was observed by Thacker et al. (60% vs. 49%).¹⁴ This situation could be related to the immunocompromised state especially during periods of neutropenia and to frequent use of antibiotics in this population.⁹ Given the characteristics of patients hospitalized in pediatric oncology units, antibiotic therapy must be initiated when the first signs of bacterial infection appear.¹⁰ Indeed, it has been previously reported that ESBL-E were more often isolated from children with previous antibiotic treatment than from those without antibiotic treatment.² Antibiotics play a major role in ESBL-E fecal carriage, and we think that it is impossible to decrease the incidence of ESBL-E without a drastic restriction of all antibiotic therapies. In children colonized by ESBL-E, we reported 5% cases of bacteremia with ESBL-E, all had hematological malignancies. However, in the same institution, Touati et al. reported that CTX-M-15 ESBL was the most commonly found in Enterobacteriaceae isolates recovered from BSI.²⁶ The authors suggested that the fecal carriage was a source of bacteremia. Moreover, Marin et al. reported that endogenous source of BSI was more common in hematological malignancies than in the solid tumor group and was mainly due to ESBL-E.⁹

We noticed in our study that death was more frequent in the ESBL-E group. That could be due to the underlying disease. However, multivariate analysis showed that both ESBL-E and hematological malignancies were independent factors associated with death. By contrast, a recent prospective observational study reported the association of multidrug resistance (MDR) with death in hematological malignancy patients.²⁷ High risk of death in cancer patients was reported, in part, due to greater delays in the introduction of appropriate therapy and the limited number of therapeutic options.^28,29

Bacterial identification by MALDI-TOF revealed that the most frequent ESBL-E isolated in this study were K. pneumoniae isolates, followed by E. coli. Similar results were reported by Zachariah et al.²⁸ Thacker et al. showed that K. pneumoniae isolates were the most frequent in children with cancer in India.¹⁴ This species was also observed in ESBL-E fecal carriage in children hospitalized with fever in Guinea-Bissau⁸ and in Gabon.³⁰

Antimicrobial sensitivity testing showed that resistance to non-β-lactam antibiotics in ESBL-E strains was high as it was previously reported by other authors.^8,13 Resistance to β-lactam antibiotics in this study was mainly due to the CTX-M-15 ESBL. This result correlates with several other studies.^10,13 In Algeria, the CTX-M-15 and CTX-M-3 were identified in both nosocomial and community isolates of various Enterobacteriaceae.^31,32

In the present study, we reported the presence of plasmid-mediated quinolone resistance (PMQR) genes in ESBL-E isolates, including aac(6′)-Ib-cr, qnrB, and qnrS. The frequency of qnr genes in the quinolone-resistant Enterobacteriaceae isolates was 26% (27/103). The qnrB gene was reported as the most prevalent over all the qnr genes.³³ In Algeria, qnrB and qnrS genes were identified for the first time, in 2008, in clinical isolates of ESBL-producing E. cloacae.³² Interestingly, numerous studies have reported the presence of PMQR in different isolates of ESBL-E,^18,32 supporting the idea that β-lactams and quinolones can act as potent coselectors for antimicrobial resistance.³³ Our results raised also the issue of potential spread of MDR bacteria or plasmids from adults to children. The phylogenetic groupings of E. coli strains revealed the presence of four main phylogroups: D, A, B1, and B2. The phylogroups D and B2 are mainly incriminated in extraintestinal infections, whereas the other phylogroups are generally considered as commensal.³⁴ This result suggests that children with cancer may be carrying virulent ESBL-E. Several reports are currently at hand on the presence and prevalence of ESBL genes in relation to the phylogenetic group in different geographical areas around the world. However, our study found no significant correlation between the CTX-M determinant and the phylogenetic group. The presence of the bla_CTX-M-15 gene in all major phylogenetic groups reflects the wide spread of this enzyme across the diversity of E. coli strains.³⁵ Morgand et al. observed (in pediatric study of ESBL E. coli infections) that phylogenetic group B2 was the most common.³⁶ Nevertheless, in our study, the group D isolates were the most frequent of ESBL E. coli. The phylogenetic groups B2 and D showed an important proportion of MDR isolates. Of note, in a study of community-acquired uropathogenic E. coli from Algeria, phylogenetic group B2 was the main group among the ESBL strains, followed by group A.³⁷ The high virulence potential of B2 strains has been largely reported based on both epidemiological studies³⁸ and animal model experiments.³⁹ This potential is largely conferred by numerous virulence factors. In contrast, A/B1 strains are less virulent, but associated with higher mortality, probably because they were more frequently found in immunocompromised patients.⁴⁰

Conclusion

In summary, ESBL-E fecal carriage was frequent in children with cancers. The results of this present study demonstrated that high prevalence of CTX-M-type ESBL-E existed in this category of children. The cancer type had a major contribution to the fecal carriage and this colonization increased the risk of death. Increasing ESBL prevalence, spreading into pediatric units, and resulting infection are a public health concern. This study provides new information on the hidden reservoir and characteristics of the target screening population, which might help to determine infection control measures to stop this growing problem.

Footnotes

Acknowledgment

The authors are grateful to J. Madoux for her contribution to this work.

Disclosure Statement

No competing financial interests exist.

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	Escherichia coli (n = 38), %	Klebsiella pneumoniae (n = 49), %	Enterobacter cloacae (n = 12), %	Klebsiella oxytoca (n = 3), %	Citrobacter braakii (n = 1), %
CTX	0	0	0	0	0
CAZ	16	6	8	33	0
FEP	8	20	0	0	0
ATM	8	6	0	0	0
FOX	97	100	NR	100	NR
TZP	92	67	75	67	100
IMP	100	100	92	100	100
ETP	100	100	92	100	100
TOB	29	26	8	0	0
GMI	29	26	33	0	0
AMK	74	61	58	33	100
CIP	45	73	50	100	100
SXT	13	10	0	0	0

	Escherichia coli (n = 38), %	Klebsiella pneumoniae (n = 49), %	Enterobacter cloacae (n = 12), %	Klebsiella oxytoca (n = 3), %	Citrobacter braakii (n = 1), %
CTX	0	0	0	0	0
CAZ	16	6	8	33	0
FEP	8	20	0	0	0
ATM	8	6	0	0	0
FOX	97	100	NR	100	NR
TZP	92	67	75	67	100
IMP	100	100	92	100	100
ETP	100	100	92	100	100
TOB	29	26	8	0	0
GMI	29	26	33	0	0
AMK	74	61	58	33	100
CIP	45	73	50	100	100
SXT	13	10	0	0	0

	Escherichia coli (n = 38), %	Klebsiella pneumoniae (n = 49), %	Enterobacter cloacae (n = 12), %	Klebsiella oxytoca (n = 3), %	Citrobacter braakii (n = 1), %
CTX	0	0	0	0	0
CAZ	16	6	8	33	0
FEP	8	20	0	0	0
ATM	8	6	0	0	0
FOX	97	100	NR	100	NR
TZP	92	67	75	67	100
IMP	100	100	92	100	100
ETP	100	100	92	100	100
TOB	29	26	8	0	0
GMI	29	26	33	0	0
AMK	74	61	58	33	100
CIP	45	73	50	100	100
SXT	13	10	0	0	0