Antimicrobial Resistance Among Gram-Negative Bacteria Isolated in the Newborn Intensive Care Unit at Abderrezak-Bouhara Hospital of Skikda,Algeria

Abstract

Background:

This study aimed to determine the epidemiology of gram-negative bacteria (GNB) isolated in the newborn intensive care unit (NICU) population, to assess their antibiotic susceptibility patterns and possible associated risk factors.

Methods:

All neonates admitted to the NICU of Abderrezak-Bouhara hospital (Skikda, Algeria) with a clinical diagnosis of neonatal infections from March to May 2019 were included in the study. The extended-spectrum β-lactamase (ESBLs), plasmidic cephalosporinase (pAmpC), and carbapenemases genes were screened by polymerase chain reaction (PCR) and sequencing. PCR amplification of oprD among carbapenem-resistant Pseudomonas aeruginosa isolates was also performed. The clonal relatedness of the ESBLs isolates was studied using multilocus sequence typing (MLST).

Results:

Among 148 clinical specimens, 36 (24.3%) GNB strains were isolated from urine (n = 22), wound (n = 8), stool (n = 3), and blood (n = 3) samples. The bacterial species identified were Escherichia coli (n = 13), Klebsiella pneumoniae (n = 5), Enterobacter cloacae (n = 3), Serratia marcescens (n = 3), Salmonella spp. (n = 3), Proteus mirabilis (n = 1), P. aeruginosa (n = 5), and Acinetobacter baumannii (n = 3). PCR and sequencing showed that eleven Enterobacterales isolates harbored the bla_CTX-M-15 gene, two E. coli isolates harbored the bla_CMY-2 gene, and three A. baumannii isolates harbored both bla_OXA-23 and bla_OXA-51 genes. Also, five strains of P. aeruginosa were found to harbor mutations in the oprD gene. MLST showed that the K. pneumoniae strains belonged to ST13 and ST189, E. coli belonged to ST69, and E. cloacae belonged to ST214. Different risk factors that could predict positive GNB cultures were found, including female sex, Apgar score <8 at 5 min of life, enteral nutrition, antibiotic use, and extended length of hospitalization.

Conclusion:

Our study highlights the importance of determining the epidemiology of pathogens causing neonatal infections, their sequence types (ST), and their antibiotic susceptibility patterns to address rapidly a correct antibiotic treatment regimen.

Introduction

Antimicrobial-resistant (AMR) gram-negative bacilli (GNB) constitute a major clinical challenge causing several health care-associated infections, particularly bacteremia (45%), pneumonia (22%), gastrointestinal infection (8%), and urinary tract infection (5%).^1,2 Systematic antibiotic misuse and overuse of these drugs in human and veterinary medicine, as well as animal production have been identified as the main factors that have contributed to the rapid increase and worldwide spread of this resistance.³ Their clinical impact is even more alarming in neonatal intensive care units (NICUs) where treatment choices are limited, contributing to morbidity and mortality.⁴ In NICU, neonates frequently receive antimicrobial agents for suspected or confirmed infections. Overall antibiotic exposure and the use of broad-spectrum cephalosporin and carbapenem agents are involved with colonization and/or infections induced by AMR-GNB.^5,6

Extended-spectrum β-lactamase-producing Enterobacterales (ESBL-PE) and carbapenem-resistant GNB represent the principal setbacks in to fight against neonatal infections and are often associated with a delay in effective antibiotics administration.⁷ Different clinical characteristics are risk factors for neonatal infections due to AMR-GNB, such as preterm birth, prolonged hospitalization, and frequent use of invasive devices and antibiotics.^8,9

The pattern of bacterial pathogens causing neonatal infections have consistently changed over time, and the emergence of AMR has complicated the problem further. In these settings, early detection of neonates colonized or infected with ESBLs or carbapenemase-producing GNB can potentially contribute to the prevention and control of late-onset infections. Only one study of ESBL-producing Klebsiella pneumoniae isolated from neonatal bloodstream infections (BSIs) were reported in NICU, Bejaia, Algeria.¹⁰ Therefore, this study aimed to determine the epidemiology of GNB isolated in the NICU population, assess current patterns of antibiotic resistance, and possible associated risk factors among neonates at the NICU of Abderrezak-Bouhara hospital, located in Algeria.

Materials and Methods

Study setting

This study was conducted at the NICU of the Abderrezak-Bouhara hospital, located in Skikda, East Algeria. This NICU is a referral center for neonates from all parts of Skikda, other neighborhood regions, and patients from private clinics with around 240 beds. Skikda city is situated on the northern shore of the Mediterranean Sea with a population of 904,195 inhabitants (According to the Algerian Census data Population statistics projections in 2022).¹¹

Study population and inclusion criteria

From March to May 2019, the study population included all neonates (0–28 days) admitted to the NICU of the Abderrezak-Bouhara hospital with a clinical diagnosis of neonatal infections at the time of admission or during their hospitalization or those with other anomalies. The neonates were examined after their admission to the NICU, and risk factors for infection were assessed. When newborns exhibit signs and symptoms, such as temperature ≤35.0°C or ≥38.0°C, metabolic acidosis, diarrhea, vomiting, or tachypnea in the absence of antibiotic medication, local clinical doctors will simultaneously request various clinical samples.

Exclusion criteria included neonates who were admitted to the NICU for 1 day's worth of diagnostic or therapeutic procedures and were later released, and neonates with the suspected viral or fungal source of infection, based on clinician judgment. Informed written consent was obtained from each study participant.

Data analysis

Demographic and clinical data were collected for each neonate and included sex, Apgar score at 5 min of life, birth weight, mode of delivery, gestational age, nutrition, antibiotic use, and length of hospitalization.

Sample collection and isolation of pathogens from clinical samples

Depending on the clinically suspected source of infection, blood (n = 23), urine (n = 97), wound (n = 16), and/or stool samples (n = 12) were collected using a convenient sampling technique, as described in the following subsections. All samples were immediately transported within hours to the clinical laboratory of Abderrezak-Bouhara hospital for analysis.

Blood specimen collection

Blood samples (2 mL of blood) were obtained and inoculated directly into culture bottles containing Brain Heart Infusion broth (Pasteur institute, Algiers, Algeria). Inoculated blood culture bottles were incubated at 37°C and inspected for 7 consecutive days. Samples positive for bacterial growth were subcultured on blood agar (Fluka, St Louis), MacConkey agar (Fluka), and Xylose Lysine Deoxycholate (XLD) agar media (Fluka).

Urine specimen collection

Urine samples were collected using the voiding stimulation method as described by Herreros Fernández et al.¹² Using a sterile calibrated loop, a urine sample was cultured on blood agar (Fluka), cysteine lactose electrolyte-deficient (Fluka), and MacConkey agar (Fluka). The number of colonies was counted after 18–24 hr of incubation at 37°C. Urinary specimens with >10⁵ colonies forming a unit per milliliter (CFU/mL) urine were considered positive.

Wound and stool sample collection

Swabs were collected aseptically with a sterile cotton-tipped swab and were inoculated onto Blood Agar and MacConkey Agar, and XLD agar media (Fluka), incubated at 37°C for 48 hr.

Bacterial strains from the cultures were identified using API Gallery and Matrix-Assisted Laser Desorption Ionization-Time of Flight mass spectrometry (MALDI-TOF MS) (bioMérieux, Mar-cyl'Étoile, France).

Antimicrobial susceptibility testing

Antimicrobial susceptibilities were determined by the use of the disk-diffusion method (Bio-Rad, Marnes La Coquette, France) on Mueller-Hinton agar according to recommendations of EUCAST 2022.¹³ The following 15 antimicrobial agents were tested: amoxicillin (25 μg), piperacillin (30 μg), ticarcillin (75 μg), cefazolin (30 μg), cefotaxime (5 μg), ceftazidime (10 μg), cefepime (30 μg), cefoxitin (30 μg), aztreonam (30 μg), ertapenem (10 μg), imipenem (10 μg), gentamicin (10 mg), amikacin (30 μg), trimethoprim-sulfamethoxazole (1.25/23.75 μg), and ciprofloxacin (5 μg).

Minimum inhibitory concentrations (MICs) of colistin were determined by microbroth dilution method (Umic^®; Biocentric, Bandol, France).¹⁴ Susceptibility patterns were interpreted according to the EUCAST 2022 guidelines.

Screening of β-lactamase-producing GNB

All isolates that exhibited reduced susceptibility and resistance to third-generation cephalosporin (3GC) were screened for ESBL-production using the double-disk synergy test.¹⁵ Escherichia coli ATCC 25922 was used for quality control strains.

The plasmidic cephalosporinase (pAmpC)-production was confirmed phenotypically using a combination of disk diffusion tests consisting of cefotaxime and ceftazidime combined with a boronic acid or cloxacillin as an inhibitor (Rosco, Taastrup, Denmark).¹⁶

The presence of carbapenemases was investigated phenotypically by the modified Carbapenem Inactivation Method (CIM) as previously described.¹⁷

Molecular characterization of resistance genes

Polymerase chain reaction (PCR) was performed to confirm the existence of ESBL genes (bla_TEM, bla_SHV, and bla_CTX-M), pAmpC-encoding genes (bla_CMY, bla_DHA, and bla_FOX), and carbapenemase genes (bla_VIM, bla_IMP, bla_KPC, bla_NDM, bla_OXA-23, bla_OXA-24, bla_OXA-48, bla_OXA-51, and bla_OXA-58) using specific primers, as described previously.^18,19

PCR amplification of oprD among carbapenem-resistant Pseudomonas aeruginosa isolates was performed using specific primers.²⁰ The amplified products were subjected to sequence analysis. DNA sequences of the oprD genes of P. aeruginosa were compared with that of reference P. aeruginosa PAO1

Analysis of clonality

Multilocus sequence typing (MLST) analysis was performed using the Institute Pasteur's MLST scheme.²¹ Seven housekeeping genes comprising for K. pneumoniae (gapA, mdh, pgi, infB, phoE, rpoB, and tonB), for E. coli (adk, gyrB, icd, mdh, fumC, purA, and recA), and for Enterobacter cloacae (dnaA, gyrB, leuS, fusA, pyrG, rplB, and rpoB) were amplified and sequenced. Moreover, sequence types (STs) were assigned by using the MLST database.

Data analysis

The comparison of variables was carried out by the chi-square test and Fisher's exact test where appropriate. Multivariate analysis was performed to determine predictors of positive GNB cultures from clinical specimens in neonates. Statistical analyses were performed using R software (version 3.3.2). Factors were considered statistically significant when the p-value was <0.05 with a corresponding 95% confidence interval.

Results

Demographic and clinical characteristics

A total of 148 neonates were enrolled. Female neonates formed the majority of the study participants (n = 81, 54.8%).

The demographic and clinical characteristics of the patients were summarized in Table 1. A total of 58 (39.1%) neonates had birth weights below 2,500 g with a mean ± standard deviation birth weight of 2,790 ± 910 g (median 2,900 g, interquartile range [IQR] 1,890–3,560 g). The median Apgar score at 5 min of life was 9 (IQR: 8–9). A total of 84 (56.8%) were delivered by cesarean section (C/S), and 82 (55.4%) had a length of stay in the NICU of <7 days.

Table 1.

The Demographic and Clinical Characteristics of the Neonates Included in This Study

Neonates characteristics	N	%/median
Mean birth weight (g)	2,790 ± 910	2,900 (IQR: 1,890–3,560)
Birth weight
<2,500 g	58	39.1
≥2,500 g	90	60.9
Sex
Male	E	45.2
Female	81	54.8
Gestation age
Full term	85	57.4
Premature	63	42.6
Perinatal history
Apgar score at 5 min of life	8.58 ± 0.91	9 (IQR: 8–9)
Cesarean section	84	56.8
Vaginal delivery	64	43.2
Nutrition
Enteral nutrition by a probe	48	32.4
Enteral feeding, exclusive breast milk	7	4.8
Enteral feeding, formula feed only	73	49.3
Enteral feeding, breast milk, and formula	20	13.5
Antibiotic treatment in NICU	135	91.2
Type of antibiotics (n = 135)
Aminopenicillins (ampicillin or amoxicillin)	135	100
Gentamicin	135	100
Amikacin	40	29.7
Third generation cephalosporins	37	27.4
Imipenem	27	20
Ciprofloxacin	11	8.1
Length of hospitalization
<7 days	82	55.4
7–15 days	48	32.5
16–30 days	18	12.1

IQR, interquartile range; NICU, newborn intensive care unit.

Most of these neonates, 135 (91.2%), received antibiotics during their hospitalization in the NICU. The common antibiotics administrated were aminopenicillins (ampicillin or amoxicillin) (100%, 135/135), gentamicin (100%, 135/135), 3GCs (27.4%, 37/135), imipenem (20%, 27/135), amikacin (29.7%, 40/135), and ciprofloxacin (8.1%, 11/135).

Concerning neonates' enteral feeding, 73 (49.3%) neonates received formula feed, 48 (32.4%) by a probe, 20 (13.5%) received both breast milk and formula, and 7 (4.8%) received exclusive breast milk.

Samples and prevalence of bacterial isolates

A total of 148 different clinical specimens collected from 148 neonates were processed during the study, of which 36 (24.3%) samples were culture-positive. Of the 36 isolates, 13 isolates were identified as E. coli, 5 K. pneumoniae, 3 E. cloacae, 3 Serratia marcescens, 3 Salmonella spp., 1 Proteus mirabilis, 5 P. aeruginosa, and 3 Acinetobacter baumannii (Table 2).

Table 2.

Distribution of Bacterial Isolates in Various Clinical Specimens Among Neonates in the Neonatal Intensive Care Unit

Clinical samples	Escherichia coli, N (%)	Klebsiella pneumonia, N (%)	Enterobacter cloacae, N (%)	Salmonella spp., N (%)	Serratia marcescens, N (%)	Proteus mirabilis, N (%)	Acinetobacter baumannii, N (%)	Pseudomonas aeruginosa, N (%)
Urine (n = 134)	13 (9.7)	5 (3.7)	0	0	0	1 (0.7)	1 (0.7)	2 (1.3)
Wound (n = 8)	0	0	3 (37.5)	0	0	0	2 (25)	3 (37.5)
Blood (n = 3)	0	0	0	0	3 (100)	0	0	0
Stool (n = 3)	0	0	0	3 (100)	0	0	0	0
Total (n = 148)	13 (8.7)	5 (3.3)	3 (2)	3 (2)	3 (2)	1 (0.6)	3 (2)	5 (3.3)

The GNB profiles revealed that 22/134 (16.4%) were isolated from the urine samples, 8/8 (100%) from the wound samples, 3/3 (100%) were from the blood samples, and 3/3 (100%) from the stool samples (Table 2).

Antibiotic susceptibility pattern of bacterial isolates

Among 36 GNB tested for multiple classes of antibiotics, all (100%, n = 36) of them were resistant to amoxicillin, piperacillin, ticarcillin, and cefazolin. They revealed 72.2% (n = 26) resistance toward cefotaxime, ceftazidime, and aztreonam, followed by cefepime (50%, n = 18) and cefoxitin (47.2%, n = 17). GNB showed a high prevalence of resistance to non-β-lactam agents, including trimethoprim-sulfamethoxazole (86.1%, n = 31), followed by gentamicin (75%, n = 27), amikacin (61.1%, n = 22), and ciprofloxacin (58.3%, n = 21). The least percentage of resistance was found toward imipenem (22.2%, n = 8) (Table 3). All isolates were susceptible to colistin (MICs <2 μg/mL).

Table 3.

Antimicrobial Resistance Profiles of Bacterial Species Among Neonates at the Neonatal Intensive Care Unit

GNB isolates (n)	Antibiotics tested
GNB isolates (n)	AMX, R (%)	PIP, R (%)	TIC, R (%)	CZ, R (%)	FOX, R (%)	CTX, R (%)	CAZ, R (%)	FEP, R (%)	ATM, R (%)	IMP, R (%)	AMK, R (%)	GEN, R (%)	CIP, R (%)	SXT, R (%)
Escherichia coli (n = 13)	13 (100)	13 (100)	13 (100)	13 (100)	2 (15.3)	4 (30.7)	4 (30.7)	2 (15.3)	4 (30.7)	0 (0)	8 (61.5)	10 (76.9)	3 (23)	11 (84.6)
Klebsiella pneumoniae (n = 5)	5 (100)	5 (100)	5 (100)	5 (100)	0 (0)	5 (100)	5 (100)	5 (100)	5 (100)	0 (0)	3 (60)	4 (80)	4 (80)	4 (80)
Enterobacter cloacae (n = 3)	3 (100)	3 (100)	3 (100)	3 (100)	3 (100)	3 (100)	3 (100)	1 (33.3)	3 (100)	0 (0)	1 (33.3)	3 (100)	2 (66.6)	2 (66.6)
Serratia marcescens (n = 3)	3 (100)	3 (100)	3 (100)	3 (100)	3 (100)	3 (100)	3 (100)	0 (0)	3 (100)	0 (0)	2 (66.6)	2 (66.6)	3 (100)	3 (100)
Salmonella spp. (n = 3)	3 (100)	3 (100)	3 (100)	3 (100)	1 (33.3)	3 (100)	3 (100)	3 (100)	3 (100)	0 (0)	1 (33.3)	1 (33.3)	1 (33.3)	3 (100)
Proteus mirabilis (n = 1)	1 (100)	1 (100)	1 (100)	1 (100)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	1 (100)	0 (0)
Pseudomonas aeruginosa (n = 5)	5 (100)	5 (100)	5 (100)	5 (100)	5 (100)	5 (100)	5 (100)	4 (80)	5 (100)	5 (100)	4 (80)	4 (80)	4 (80)	5 (100)
Acinetobacter baumannii (n = 3)	3 (100)	3 (100)	3 (100)	3 (100)	3 (100)	3 (100)	3 (100)	3 (100)	3 (100)	3 (100)	3 (100)	3 (100)	3 (100)	3 (100)
Total (n = 36)	36 (100)	36 (100)	36 (100)	36 (100)	17 (47.2%)	26 (72.2%)	26 (72.2%)	18 (50%)	26 (72.2%)	8 (22.2%)	22 (61.1%)	27 (75%)	21 (58.3%)	31 (86.1%)

AMX, amoxicillin; AMK, amikacin; ATM, aztreonam; CAZ, ceftazidime; CEP, cefepime; CIP, ciprofloxacin; CTX, cefotaxime; CZ, cefazolin; FOX, cefoxitin; GEN, gentamicin; GNB, Gram-negative bacilli; IMP, imipenem; n, total number; PIP, piperacillin; R%, percent of bacterial isolates resistant to antimicrobial agents; SXT, trimethoprim-sulfamethoxazole; TIC, ticarcillin.

The double-disc synergy test was positive for 11 Enterobacterales isolates (7.4%), whereas the double-disc synergy test with cloxacillin was positive for 2 Enterobacterales isolates (1.3%). The CIM test was positive for eight GNB isolates.

Prevalence of β-lactamase-producing GNB strains

In total, 11/148 clinical samples showed an ESBL phenotype (7.4%), of which 7/134 (5.2%) were isolated from urine samples, 3/3 (100%) from stool samples, and 1/8 (12.5%) from wound samples. These ESBL-producing Enterobacterales were distributed as follows: 5/36 (13.8%) of K. pneumoniae, 3/36 (8.3%) of Salmonella spp., 2/36 (5.5%) of E. coli, and 1/36 (2.7%) of E. cloacae.

In addition to ESBL producers, two (2/148, 1.3%) E. coli strains isolated from urine samples were found to be pAmpC producers.

The proportion of carbapenemase-resistant was 8/148 (5.4%) among P. aeruginosa (n = 5) and A. baumannii (n = 3) species. The proportion of carbapenemase-resistant was 3/134 (2.2%) and 5/8 (62.5%) from urine and wound samples, respectively.

Genomic analysis of GNB isolates

Among the 11 ESBL producers, all of them carried the bla_CTX-M-15 gene. In addition, the two pAmpC-producing E. coli isolates harbored the bla_CMY-2 gene. Carbapenemase-encoding bla_OXA-23 and bla_OXA-51 genes were detected in the three A. baumannii strains (Table 4).

Table 4.

Phenotypic and Genotypic Features of the 11 Extended-Spectrum β-Lactamases, 2 Plasmidic Cephalosporinase, and 8 Carbapenemase-Producing Strains Isolated from Neonates

Code	Sample origin	Sex	Age (days)	Weight (g)	Gestation age	Bacterial species	Antibiotic resistance pattern	Resistance mechanism	Sequence type
P1	Urine	Female	13	3,300	Full term	Escherichia coli	AMX, PIP, TIC, CZ, CTX, CAZ, FEP, ATM, AMK, GEN, SXT	CTX-M-15	69
P2	Urine	Female	18	1,500	Premature	E. coli	AMX, PIP, TIC, CZ, CTX, CAZ, FEP, ATM, AMK, GEN, CIP	CTX-M-15	69
P23	Urine	Female	17	3,580	Full term	Klebsiella pneumoniae	AMX, PIP, TIC, CZ, CTX, CAZ, FEP, ATM, CIP, SXT	CTX-M-15	13
P24	Urine	Female	12	1,700	Premature	K. pneumoniae	AMX, PIP, TIC, CZ, CTX, CAZ, FEP, ATM, AMK, GEN, SXT	CTX-M-15	189
P25	Urine	Female	28	4,500	Full term	K. pneumoniae	AMX, PIP, TIC, CZ, CTX, CAZ, FEP, ATM, GEN, CIP, SXT	CTX-M-15	13
P35	Urine	Female	28	1,530	Premature	K. pneumoniae	AMX, PIP, TIC, CZ, CTX, CAZ, FEP, ATM, AMK, GEN, CIP, SXT	CTX-M-15	189
P36	Urine	Female	14	3,500	Full term	K. pneumoniae	AMX, PIP, TIC, CZ, CTX, CAZ, FEP, ATM, AMK, GEN, CIP	CTX-M-15	13
P43	Wound	Female	9	4,000	Full term	Enterobacter cloacae	AMX, PIP, TIC, CZ, CTX, CAZ, FEP, ATM, FOX, AMK, GEN, CIP, SXT	CTX-M-15	214
P49	Stool	Female	26	3,630	Full term	Salmonella spp.	AMX, PIP, TIC, CZ, CTX, CAZ, FEP, ATM, AMK, GEN, SXT	CTX-M-15	ND
P50	Stool	Female	10	1,310	Premature	Salmonella spp.	AMX, PIP, TIC, CZ, CTX, CAZ, FEP, ATM, AMK, CIP, SXT	CTX-M-15	ND
P51	Stool	Male	14	3,630	Full term	Salmonella spp.	AMX, PIP, TIC, CZ, CTX, CAZ, FEP, ATM, FOX, SXT	CTX-M-15	ND
P3	Urine	Female	26	2,300	Premature	E. coli	AMX, PIP, TIC, CZ, CTX, CAZ, FEP, ATM, FOX, AMK, GEN, SXT	CMY-2	ND
P5	Urine	Female	26	3,700	Full term	E. coli	AMX, PIP, TIC, CZ, CTX, CAZ, FEP, ATM, FOX, AMK, GEN, SXT	CMY-2	ND
1	Wound	Male	17	1,700	Premature	Acinetobacter baumannii	AMX, PIP, TIC, CZ, CTX, CAZ, FEP, ATM, FOX, IMP, AMK, GEN, CIP, SXT	OXA-23	ND
2	Wound	Female	15	2,700	Premature	A. baumannii	AMX, PIP, TIC, CZ, CTX, CAZ, FEP, ATM, FOX, IMP, AMK, GEN, CIP, SXT	OXA-23	ND
4’	Urine	Female	10	2,200	Premature	A. baumannii	AMX, PIP, TIC, CZ, CTX, CAZ, FEP, ATM, FOX, IMP, AMK, GEN, CIP, SXT	OXA-23	ND
9	Wound	Male	23	3,980	Full term	Pseudomonas aeruginosa	AMX, PIP, TIC, CZ, CTX, CAZ, FEP, ATM, FOX, IMP, AMK, CIP, SXT	OprD loss	ND
10	Urine	Female	23	4,300	Full term	P. aeruginosa	AMX, PIP, TIC, CZ, CTX, CAZ, FEP, ATM, FOX, IMP, GEN, CIP, SXT	OprD loss	ND
11	Wound	Female	15	2,640	Premature	P. aeruginosa	AMX, PIP, TIC, CZ, CTX, CAZ, ATM, FOX, IMP, AMK, GEN, CIP, SXT	OprD loss	ND
14	Urine	Female	14	1,600	Premature	P. aeruginosa	AMX, PIP, TIC, CZ, CTX, CAZ, FEP, FOX, IMP, AMK, GEN, SXT	OprD loss	ND
13	Wound	Male	19	1,800	Premature	P. aeruginosa	AMX, PIP, TIC, CZ, CTX, CAZ, FEP, ATM, FOX, IMP, AMK, GEN, CIP, SXT	OprD loss	ND

ND, not determined.

The oprD typing results showed that five P. aeruginosa isolates harbored the same missense mutation C748T in the oprD gene (Table 4).

Multilocus sequence typing

MLST demonstrated the presence of two STs among the K. pneumoniae isolates, including ST13 (n = 3) and ST189 (n = 2), whereas only one clone (ST69) was identified for two isolates of E. coli. The E. cloacae isolate belonged to ST214 (Table 4).

Statistical analysis

We investigated different factors that could predict positive GNB cultures isolated from neonates. Female sex, Apgar score <8 at 5 min of life, enteral nutrition by a probe, extended length of hospitalization, and antibiotics use (aminopenicillins, gentamicin, amikacin, 3GC, imipenem, and ciprofloxacin) were significantly associated with positive GNB cultures isolated from neonates (p < 0.001) (Table 5).

Table 5.

Predictors of Positive Gram-Negative Bacteria Cultures and Negative Gram-Negative Bacteria Cultures Isolated from Neonatal Clinical Samples

Neonates characteristics	Negative GNB cultures (n = 112)	Positive GNB cultures (n = 36)	p
Mean birth weight (kg)	2,850 ± 880	2,590 ± 1,000	0.15
Birth weight
<2,500 g	41 (36.7)	17 (47.2)	0.26
≥2,500 g	71 (63.3)	19 (52.8)	0.26
Sex
Male	57 (50.9)	10 (27.8)	0.015
Female	55 (49.1)	26 (72.2)	0.015
Gestation age
Full term	68 (60.8)	17 (47.2)	0.15
Premature	44 (39.2)	19 (52.8)	0.15
Perinatal history
Apgar score at 5 min of life	8.70 ± 0.781	8.22 ± 1.20	<0.01
Cesarean section	60 (53.6)	24 (66.7)	0.17
Vaginal delivery	52 (46.4)	12 (33.3)	0.17
Nutrition
Enteral nutrition by a probe	30 (26.8)	18 (50)	<0.01
Enteral feeding, exclusive breast milk	4 (3.6)	3 (8.3)
Enteral feeding, formula feed only	64 (57.1)	9 (25)
Enteral feeding, breast milk, and formula	14 (12.5)	6 (16.7)
Antibiotic treatment in NICU
Yes	99 (88.3)	36 (100)	0.038
No	13 (11.7)	0 (0)	0.038
Type of antibiotics
Aminopenicillins (ampicillin or amoxicillin)
Yes	99 (88.3)	36 (100)	0.038
No	13 (11.7)	0 (0)	0.038
Gentamicin
Yes	99 (88.3)	36 (100)	0.038
No	13 (11.7)	0 (0)	0.038
Third generation cephalosporins
Yes	9 (8)	28 (77.8)	<0.001
No	103 (92)	8 (22.2)	<0.001
Imipenem
Yes	4 (3.6)	23 (63.9)	<0.001
No	108 (96.4)	13 (36.1)	<0.001
Ciprofloxacin
Yes	0 (0)	11 (30.6)	<0.001
No	112 (100)	25 (69.4)	<0.001
Amikacin
Yes	13 (11.7)	27 (75)	<0.001
No	99 (88.3)	9 (25)	<0.001
Length of hospitalization
<7 days	78 (69.7%)	4 (11.1%)	<0.001
7–15 days	27 (24.1%)	21 (58.3%)
16–30 days	7 (6.2%)	11 (30.6%)

Discussion

In the present study, we screened 148 clinical samples at the NICU of Abderrezak-Bouhara hospital (Skikda, Algeria) for the presence of GNB isolates. We found an overall prevalence of 24.3%. These GNB isolates were resistant to amoxicillin (100%), gentamicin (75%), and 3GC (72.2%), consistent with recent findings in Algeria and across sub-Saharan Africa.^10,22 World Health Organization (WHO) guidelines stipulate aminopenicillin (ampicillin or amoxicillin) in combination with gentamicin as the first line of empirical treatment for neonatal infections and 3GC as the second line of treatment.²³

Alarmingly, AMR was overly present in our isolates, including broad-spectrum antibiotics such as carbapenems (22.2%), limiting the therapeutic choices of neonates. In addition, we noticed a high rate of resistance to ciprofloxacin (58.3%) and trimethoprim-sulfamethoxazole (86.1%) antibiotics not habitually used in neonates but frequently approved for treating infections among adult patients in Algeria. This underlines the urgency for more appropriate use of antibiotics across all age groups within a population.

The predominant resistance mechanism in our study is the production of ESBLs; we found that 7.4% of screened neonates were infected with ESBLs during their hospitalization at NICU, which was lower than the prevalence rate found in Algeria from neonatal BSIs (8.2%).¹⁰ This prevalence of ESBLs could be attributed to the prescription of 3GC as a second-line treatment option in neonates. However, several authors have reported the high prevalence of ESBLs in adult infections in Algerian hospitals (19–47%).^24–29

The reasons for a lower prevalence rate in neonates compared to adult patients may be attributed to the quality of health care, aseptic technique practice, and standard diagnostic setup. The CTX-M type was the only ESBL recovered in our isolates, which was the most common ESBL isolated in Algeria.³⁰ The plasmidic bla_AmpC gene of E. coli isolates was identified as bla_CMY-2, this allele was detected in clinical isolates from some studies conducted in Algerian hospitals.^31–34

In this study, MLST showed that the ESBL-positive K. pneumoniae isolates belonged to ST13 and ST189. The ST13 contributes to the spread of carbapenemase genes from different ecological niches in Algeria,^35,36 whereas the ST189 has been reported in Algeria and identified in CTX-M-15-producing K. pneumoniae strains isolated from neonatal bloodstream infections.¹⁰ The ESBL-producing E. coli isolates were assigned to ST69. This clone has spread worldwide,³⁷ and has already been detected in CTX-M-15-producing E. coli from the wild bird in Algeria.³⁸ The E. cloacae ST214 clone recovered in our study has also been reported in ESBL-producers in Algerian hospitals.³⁹

The prevalence rate of carbapenemase-producing GNB among hospitalized neonates was 5.4%, while no resistance conferring to carbapenemase was reported in the study conducted by Mairi et al, among neonatal BSIs in Algeria.¹⁰ These variations may be due to the high utilization of carbapenems in our study area and this antibiotic is reserved for the treatment of infections caused by multidrug-resistant GNB, especially β-lactamases carrying genes for ESBLs and derepressed AmpC.⁴⁰

In the current study, we reported three A. baumannii isolates producing the bla_OXA-23 gene. The emergence and spread of the A. baumannii-producing bla_OXA-23 gene have been reported from clinical samples^41–46 and inanimate surfaces in several Algerian hospitals.^47,48 Additional screening for the oprD gene in P. aeruginosa demonstrated that five isolates harbored missense mutations (C748T) in this gene leading to truncation at amino acid 249. Unfortunately, we were unable to determine if these five strains were clonally related. Inactivating mutations in OprD has been documented worldwide to confer resistance to carbapenems.⁴⁹ In Algeria, several reports on P. aeruginosa harboring different mutations in the oprD gene in adult infections have been published.^43,50–52

Factors that predispose to AMR-GNB infections in the neonatal period include prior antibiotic use (aminopenicillin, 3GC, carbapenem, ciprofloxacin, and aminoglycoside exposure), Apgar score <8 at 5 min of life, enteral nutrition by a probe, and female sex. Several studies have reported Apgar <8 at 5 min of life,^10,53,54 antibiotic use,^10,55–58 enteral nutrition by a probe,¹⁰ female sex,⁶ and the extended length of stay in NICU¹⁰ significantly associated with AMR-GNB neonatal infections.

In all, this study indicated a high prevalence of AMR-GNB coharboring different antibiotic resistance mechanisms (ESBLs, pAmpC, and carbapenem producers) from neonates in Algeria. Appropriate and adequate antibiotic use will enable us to counteract antimicrobial resistance in bacterial pathogens, and control of the hygiene of the NICU is needed to prevent the dissemination of these pathogens.

Footnotes

Acknowledgment

We thank the “Direction Générale de la Recherche Scientifique et du développement technologique (DGRSDT)” of the Algerian Ministry of Higher Education and Scientific Research.

Authors' Contributions

A.L.: collected the data, analyzed them, and wrote the article. S.B.: analyzed the data. D.G.K.: corrected the article. A.T.: analyzed the data and corrected the article. All authors have read and approved the final version of this article.

Ethical Approval

This study protocol was approved by the local Ethics Committee of Elmeki Hospital. It was performed in line with the principles of the Declaration of Helsinki. Parents were informed about the purposes of the research study, procedures, risks, benefits, confidentiality, and rights of participants. Then, written informed consent for study participation and to publish the findings was obtained from parents on behalf of their respective children. All patients' information was kept anonymous and confidential using study codes.

Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

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