First Detection of VIM-4-Producing Pseudomonas aeruginosa and OXA-48-Producing Klebsiella pneumoniae in Northeastern (Annaba,Skikda) Algeria

Abstract

Purpose:

The aim of the study was to investigate the prevalence and molecular support of carbapenem resistance in gram-negative bacilli clinical isolates collected between March 2013 and March 2015 in three cities (Annaba, Skikda, and Guelma) in northeastern Algeria.

Results:

One hundred eighty-six isolates were identified as Enterobacteriaceae (161), Pseudomonas aeruginosa (18), and Acinetobacter baumannii (7). Thirty-six of 186 (19.3%) were resistant to carbapenems. Among them, 11 harbored carbapenemase genes, including bla_OXA-48 (2 Klebsiella pneumoniae), bla_VIM-4 (2 P. aeruginosa), bla_NDM-1 (2 A. baumannii), and bla_OXA-23 (5 A. baumannii). In addition, other β-lactamases were detected: bla_{CTX-M-(15/66/139)}, bla_{SHV-(28/85/1/133)}, and bla_TEM-1. All imipenem-resistant P. aeruginosa displayed OprD mutations. Multilocus sequence typing demonstrated the presence of ST 404 and ST 219 in K. pneumoniae, ST 2 and ST 85 in A. baumannii, and ST (244, 1076, 241, 227, and 233) in P. aeruginosa.

Conclusion:

In this study, we report the first detection of P. aeruginosa ST 1076 harboring the bla_VIM-4 gene in African countries in two cities (Annaba and Skikda) in northeastern Algeria. Additionally, we report the first detection of bla_OXA-48 in K. pneumoniae ST 404 and ST 219 in Algerian cities (Annaba and Skikda).

Introduction

Rapid evolution of antibiotic resistance in bacteria is currently a worrying phenomenon in developing countries, particularly in some African countries, where antibiotic-resistant pathogens may have higher prevalence.¹ Algeria is a country in northern Africa, where recent antibiotic resistance data indicate a worrying situation. Indeed, the past 10 years have been marked by the emergence and spread of new resistance genes, particularly in the north of the country. Resistance has been observed mainly among different species of Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae), Pseudomonas aeruginosa, and Acinetobacter baumannii.²

Carbapenems are a β-lactam group of drugs that are often used as antibiotics of last resort to treat infections resulting from multidrug-resistant gram-negative bacilli. Carbapenems are restricted to hospital use and are mainly prescribed for the treatment of nosocomial infections. However, in recent years, this scenario has changed, with the emergence of carbapenem-resistant bacteria both in nonfermentative (A. baumannii and P. aeruginosa) and fermentative (Enterobacteriaceae) gram-negative bacilli.³

Carbapenemase enzymes have been reported extensively in Enterobacteriaceae around the world.

Currently, carbapenemases in Enterobacteriaceae are mainly found in K. pneumoniae and, to a much lesser extent, in E. coli and other Enterobacterial species, with a higher prevalence in southern Europe and Asia than in other parts of the word. Class D β-lactamases, also known as bla_OXAs for oxacillinases, have been detected in many gram-negative bacteria, including Enterobacteriaceae. The bla_OXA-48 gene was initially identified in a K. pneumoniae isolate from Turkey in 2001.³ Since then, the occurrence of bla_OXA-48 producers has been reported as the source of nosocomial outbreaks in many parts of the world, notably in Mediterranean countries: Croatia, Egypt, France, Greece, Israel, Italy, Lebanon, Libya, Slovenia, Spain, Tunisia, Morocco, and Turkey.³ Moreover, among the increasingly reported and commonly identified multi- or even pan-drug-resistant bacteria, P. aeruginosa holds an important place. Several types of metallo-β-lactamases (MBLs) have been described around the world in P. aeruginosa isolates (IMP, VIM, SPM, GIM, SIM, AIM, FIM, and NDM).⁴ The most frequent of the MBLs are the VIM types. These have been identified in carbapenem-resistant isolates of P. aeruginosa in European countries with coastlines on the Mediterranean basin: Italy, France, Greece, Spain, Croatia, and Turkey and from African countries: Tunisia, Kenya, Libya, South Africa,⁴ and Lebanon.⁵ The main porin for uptake of carbapenems in P. aeruginosa is the outer membrane protein, OprD. Inactivating mutations in the OprD gene represent the most common molecular mechanism known for conferring resistance to carbapenems.⁵

In Algeria, from the beginning of 2010, carbapenemases of the Ambler class D (OXA-23 and OXA-58) type or class B type, including VIM-19 enzyme, were considered to be the first carbapenemases detected in A. baumannii^6,7 and Enterobacteriaceae,⁸ respectively. Following that other types of carbapenemase classes, B (VIM-2, NDM-1, and NDM-5 enzymes), A (KPC enzymes), and D (OXA-23, OXA-24, and OXA-48 enzymes), were reported, with some of them becoming endemic in Algeria (e.g., OXA-23 and NDM-1 in A. baumannii).^7,9

The objectives of the present study were therefore to investigate the prevalence and molecular support of carbapenem resistance in gram-negative bacilli from clinical isolates collected between March 2013 and March 2015 from patients in three cities (Annaba, Skikda, and Guelma) in northeastern Algeria.

Materials and Methods

Data collection

A total of 186 nonduplicated gram-negative bacilli strains were isolated from patients hospitalized between March 2013 and March 2015 in three cities (Annaba, Skikda, and Guelma) in northeastern Algeria. These strains were identified using API 20E (Enterobacteriaceae) and API 20NE (P. aeruginosa and A. baumannii) identification systems (bioMérieux) and were confirmed using a matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) method (Microflex; Bruker Daltonics) as previously described.¹⁰

Antibiotic susceptibility testing

Antimicrobial drug susceptibility was determined using the disk diffusion method on Mueller–Hinton (MH) agar according to the Antibiogram Committee of the Société Française de Microbiologie (CA-SFM) (www.sfm-microbiologie.org). Seventeen antibiotics were tested: amoxicillin, amoxicillin/clavulanic acid, ticarcillin/clavulanic acid, cefoxitin, cefotaxime, aztreonam, ceftriaxone, ceftazidime, imipenem, ertapenem, trimethoprim/sulfamethoxazole, amikacin, gentamicin, ciprofloxacin, fosfomycin, rifampicin, and colistin. The minimum inhibitory concentrations for imipenem were determined using the Etest method (ABbiodisk). Interpretations were made according to CA-SFM breakpoints.

Phenotypic detection of carbapenemases

Phenotypic detection of carbapenemase production was performed using the modified Hodge test (MHT), the EDTA test, and modified Carba NP test (MCNP), as previously described.^11,12

Detection of antibiotic resistance genes

Gram-negative bacilli isolates were screened using standard and real-time PCR for the following carbapenem-hydrolyzing enzyme-encoding genes: bla_OXA-48,¹³ bla_VIM,⁵ bla_NDM,¹⁴ bla_KPC,¹⁵ bla_IMP,⁵ bla_OXA-58,¹⁶ bla_OXA-24,¹⁶ bla_OXA-23,¹⁶ and other β-lactamases: (bla_TEM, bla_SHV, bla_CTXM)¹⁶ and bla_OprD.⁵

Sequencing of resistance genes

All amplified products obtained were sequenced to validate their identities. Both strands of the purified amplicons were sequenced using Big Dye terminator chemistry on an ABI 3130XL automated sequencer (Applied Biosystems). The nucleotide and deduced protein sequences were analyzed using the ARG-ANNOT (Antibiotic Resistance Gene-ANNOTation).¹⁷ PCR products of OprD were fully sequenced as described above, and the resulting sequences were compared with the PAO1 reference strain sequence.

Genotyping clinical isolates

The epidemiology of K. pneumoniae, P. aeruginosa, and A. baumannii carbapenemase production was studied by multilocus sequence typing (MLST) as described.^4,18 Isolates were attributed a sequence-type (ST) number according to the allelic profiles available on the Institut Pasteur's MLST website (www.pasteur.fr/mlst) and PubMLST (www.pubmlst.org).

Results

Between March 2013 and March 2015, 186 gram-negative bacilli were collected from three hospitals in northeastern Algeria, including Annaba, n = 114 (61.2%), Skikda, n = 47 (25.2%), and Guelma, n = 25 (13.4%). Identification using both API20NE or API20E (identification system) and MALDI-TOF MS led to the identification of 161 (86.5%) Enterobacteriaceae (54 E. coli [29%], 55 Klebsiella spp. [29.5%], 17 Enterobacter spp. [9.1%], 10 Serratia spp. [5.3%], 12 Proteus spp. [6.4%], 2 Citrobacter spp. [1.07%], 8 Morganella morganii [4.3%], and 3 Providencia spp. [1.6%]) as well as 25 (13.4%) nonfermenter species (18 P. aeruginosa [9.6%] and 7 A. baumannii [3.76%]). The strains were isolated from wounds (61.2%), urine (35.4%), bloodstream infections (2.1%), and pleural fluid (1%). These samples were recovered from various hospital units (internal medicine, general surgery, pediatrics, urology, traumatology, operating rooms, burns unit, accident and emergency, and neonatology).

The Enterobacteriaceae showed a high degree of resistance to amoxicillin, amoxicillin/clavulanic acid, cefotaxime, and rifampicin. All strains were resistant to at least one aminoglycoside, including gentamicin. A lower rate of resistance was observed for ertapenem and colistin (Table 1).

Table 1.

Antimicrobial Susceptibility in Gram-Negative Bacilli Clinical Isolates

					Resistance (%)
	Enterobacteriaceae N = 161								Gram-negative bacilli nonfermenters N = 25
Antibiotics	1	2	3	4	5	6	7	8	9	10
Amoxicillin	74.5	87.5	93.3	77.7	66.6	100	100	100	100	100
Amoxicillin/clavulanic acid	65.4	70	93.3	77.7	50	50	100	100	100	100
Ticarcillin/clavulanic acid	67.2	75	80	22.2	25	0	0	33.3	50	100
Cefoxitin	29	30	80	33.3	25	0	0	0	100	100
Cefotaxime	38.1	75	40	66.6	33.3	50	16.6	66.6	100	100
Aztreonam	38.1	62.5	33.3	44.4	8.3	0	0	0	33.3	71.8
Ceftriaxone	38.1	80	46.6	33.3	25	0	0	33.3	/	/
Ceftazidime	/	/	/	/	/	/	/	/	44.4	100
Imipenem	0	0	0	0	0	0	0	0	50	100
Ertapenem	12.7	20	46.6	22.2	0	0	0	0	100	100
Amikacin	9	37.5	26.6	0	8.3	0	0	0	44.4	100
Gentamicin	25.4	57.5	26.6	0	8.3	0	0	33.3	50	100
Ciprofloxacin	27.2	37.5	6.6	0	0	0	33.3	66.6	38.8	100
Trimethoprim/sulfamethoxazole	56.3	72.5	26.6	22.2	83.3	0	16.6	66.6	100	100
Fosfomycine	0	52.5	26.6	11.1	8.3	0	33.3	66.6	100	100
Rifampicin	63.6	87.5	93.3	44.4	25	100	0	33.3	88.9	100
Colistin	0	0	0	100	100	0	0	0	0	0

1: Escherichia coli, 2: Klebsiella spp., 3: Enterobacter spp., 4: Serratia spp., 5: Proteus spp., 6: Citrobacter spp.,7: Morganella morganii, 8: Providencia spp. 9: Pseudomonas aeruginosa, 10: Acinetobacter baumannii.

Of 161 Enterobacteriaceae, 20 (12.4%) were screened for carbapenemase based on their reduced susceptibility to ertapenem (Table 2) and were included in the molecular biology study. Two K. pneumoniae isolates were positive by MHT and MCNP, bla_OXA-48 was detected in these two isolates with ST 404 and ST 219. Neither bla_KPC nor bla_NDM gene was detected. A wide range of β-lactamases was found, and further analysis of bla_TEM and bla_SHV indicated the presence of bla_TEM-1 (n = 9), bla_SHV-28, bla_SHV-85, bla_SHV-1, and bla_SHV-133 in only four isolates. The bla_CTX-M-15 gene was detected (n = 15) and two isolates carried the bla_CTX-M-139 gene and one K. pneumoniae carried the bla_CTX-M-66 gene (Table 2).

Table 2.

Phenotypic and Genotypic Features of ESBLs and Carbapenemase-Producing Enterobacteriaceae Clinical Isolates

	Isolate	Location	Date of isolation	Ward	Type of swebs	IMP	ETP	MHT	MCNP	ESBL	Carbapenemase	MLST
Enterobacter cloacae	12	Annaba	07–2014	Bloc	Wound	S	R	+	−	CTX-M-15	−	NT
	7	Annaba	04–2014	Bloc	Wound	S	R	−	−	−	−	NT
	3	Skikda	07–2013	Bloc	Wound	S	R	−	−	CTX-M-15, TEM-1	−	NT
	6	Skikda	07–2013	Neonatology	Urine	S	R	+	−	CTX-M-15, TEM-1	−	NT
	11	Skikda	08–2013	General surgery	Wound	S	R	−	−	CTX-M-15, TEM-1	−	NT
	1	Skikda	06–2013	Traumatology	Urine	S	R	−	−	CTX-M-15, TEM-1	−	NT
	5	Guelma	05–2013	Internal medicine	Urine	S	R	−	−	CTX-M-15	−	NT
Klebsiella pneumoniae	24	Skikda	03–2015	Bloc	Wound	S	R	+	+	CTXM-15, SHV-133	OXA-48	ST 404
	58	Annaba	01–2015	Burn unit	Wound	S	R	+	+	CTXM-15, SHV-85	OXA-48	ST 219
	56	Skikda	03–2015	Pediatric	Blood culture	S	R	−	−	CTX-M-15	−	NT
	11	Annaba	07–2013	Pediatric	Urine	S	R	−	−	CTX-M-139, TEM-1, SHV-28	−	NT
	05	Skikda	07–2013	Internal medicine	Urine	S	R	+	−	CTX-M-15, TEM-1, SHV-1	−	NT
	10	Annaba	08–2013	Neonatology	Urine	S	R	−	−	CTX-M-66, TEM-1	−	NT
E. coli	24	Skikda	06–2014	Neonatology	Urine	S	R	−	−	CTX-M-15	−	NT
	25	Annaba	11–2014	Internal medicine	Wound	S	R	−	−	TEM-1	−	NT
	51	Guelma	05–2013	Internal medicine	Urine	S	R	−	−	CTX-M-15	−	NT
	60	Guelma	05–2013	Internal medicine	Urine	S	R	−	−	CTX-M-15, TEM-1	−	NT
	6	Skikda	10–2013	Neonatology	Urine	S	I	−	−	CTX-M-139	−	NT
Serratiamar	4	Annaba	07–2013	Internal medicine	Wound	S	R	−	−	CTX-M-15	−	NT
	8	Annaba	05–2013	Pediatric	Wound	S	I	+	−	CTX-M-15	−	NT

ESBL, extended spectrum beta-lactamase; ETP, ertapenem; IMP, imipenem; MCNP, modified Carba NP; MHT, modified Hodge test; MIC, minimum inhibitory concentration; MLST, multilocus sequence typing; NT, not tested.

In terms of the nonfermenter bacilli, the results of antibiotic susceptibility testing revealed that the isolates were resistant to almost all antibiotics, including β-lactams, aminoglycosides, and fluoroquinolones. In addition, 100% of A. baumannii and 50% of P. aeruginosa isolates were resistant to imipenem. All isolates were susceptible to colistin except Serratia and Proteus species (Table 1).

Of the nine imipenem-resistant P. aeruginosa isolates, β-lactamase activity was inhibited by the EDTA in only two, and PCR revealed a bla_VIM-4 gene in these isolates. None of the strains contained bla_IMP, bla_NDM, bla_OXA-58, bla_OXA-24, bla_OX-23, bla_TEM, bla_SHV, and bla_CTX-M genes (Table 3). Due to various mutations, all carbapenem-resistant P. aeruginosa isolates had a modification in the amino acid sequence of the OprD protein based on comparison with the PAO1 reference. Indeed, all isolates had modifications in their OprD gene sequence with insertion sequences at different positions, leading to stop codons and causing mutations in the OprD gene sequences. Based on the mutations in OprD, the P. aeruginosa isolates could be classified into five groups (Table 3).

Table 3.

Phenotypic and Genotypic Features of the 16 Imipenem-Resistant Pseudomonas aeruginosa and Acinetobacter baumannii Clinical Isolates Producing ESBLs and Carbapenemases

	Isolates	Locations	Date of isolation	Ward	Type of swebs	IMP	MIC IMP mg/L	MHT	EDTA test	MCNP	ESBL	Carbapenemase	Mutation OPRD	MLST
P. aeruginosa	1	Annaba	03–2014	Burn unit	Wound	R	>32	−	−	−	−	−	G1(TAG_40,137)	ST 244
	ALG-2	Skikda	03–2014	Pediatric	Wound	R	>32	+	+	+	−	VIM-4	G2(TGA_60,190)	ST 1076
	3	Annaba	12–2014	Burn unit	Wound	R	>32	−	−	−	−	−	G3(TAG₁₃₄)	ST 241
	4	Annaba	12–2014	Burn unit	Wound	R	>32	−	−	−	−	−	G4(TGA₆₄)	ST 227
	5	Annaba	12–2014	Burn unit	Wound	R	>32	−	−	−	−	−	G5(TGA₁₈₀)	ST 233
	11	Annaba	12–2014	Burn unit	Wound	R	>32	−	−	−	−	−	G1(TAG_40,137)	ST 244
	ALG-19	Annaba	12–2014	Burn unit	Wound	R	>32	+	+	+	−	VIM-4	G2(TGA_60,190)	ST 1076
	20	Annaba	12–2014	Burn unit	Wound	R	>32	−	−	−	−	−	G1(TAG_40,137)	ST 244
	23	Annaba	12–2014	Burn unit	Wound	R	>32	−	−	−	−	−	G2(TGA_60,190)	ST 1076
A. baumannii	1	Annaba	03–2013	General surgery	Wound	R	>32	+	−	+	TEM-1	OXA-23	/	ST 2
	2	Skikda	06–2013	Internal medicine	Plural Fluid	R	>32	+	+	+	−	NDM-1	/	ST 85
	4	Annaba	12–2014	Bloc	Wound	R	>32	+	−	+	TEM-1	OXA-23	/	ST 2
	5	Annaba	12–2013	Internal medicine	Wound	R	>32	+	−	+	TEM-1	OXA-23	/	ST 2
	6	Annaba	02–2014	Internal medicine	Wound	R	>32	+	−	+	TEM-1	OXA-23	/	ST 2
	7	Annaba	03–2013	Pediatric	Wound	R	>32	+	+	+	−	NDM-1	/	ST 85
	8	Annaba	03–2014	Bloc	Wound	R	>32	+	−	+	TEM-1	OXA-23	/	ST 2

G1: insertion of G in nucleotide positions 40 and 137 leading to the premature stop codon TAG in oprD, resulting in a truncated polypeptide of 200 amino acid residues, G2: insertion of C in nucleotide positions 60 and 190 leading to the premature stop codon TGA in oprD, resulting in a truncated polypeptide of 64 amino acid residues, G3: insertion of G in nucleotide positions 134 leading to the premature stop codon TAG in oprD, resulting in a truncated polypeptide of 153 amino acid residues, G4: insertion of C in nucleotide positions 64 leading to the premature stop codon TGA in oprD, resulting in a truncated polypeptide of 89 amino acid residues, G5: insertion of C in nucleotide positions 180 leading to the premature stop codon TGA in oprD, resulting in a truncated polypeptide of 222 amino acid residues.

The MLST of P. aeruginosa revealed the presence of multiple clones (244, 1076, 241, 227, and 233) among the imipenem-resistant strains and which comprised strains from the hospitals of Annaba and Skikda with identical STs and identical OprD sequences. The compositions of these groups were as follows: G1, G2, G3, G4, and G5 were isolated from Annaba and consisted of ST 244, 1076, 241, 227, and 233, respectively. The G2 that was isolated from the Skikda Hospital consisted of ST 1076 (Table 3).

All imipenem-resistant A. baumannii tested were positive using MHT and MCNP and two of them tested were positive with the EDTA test. Carbapenemase genes were present in most A. baumannii, including bla_OXA-23 (n = 5), and only two isolates were positive for the bla_NDM-1 gene. None of the strains contained the bla_OXA-58, bla_OXA-24, bla_VIM, and bla_IMP genes. Furthermore, genotypic characterization of extended spectrum beta-lactamase (ESBLs) revealed the identification of the bla_TEM-1 gene in five strains. The clonal relationship of A. baumannii isolates based on MLST analysis showed the presence of ST 2 and 85 (Table 3).

Discussion

Over the past 10 years, we have seen a significant increase in antibiotic resistance in gram-negative bacilli in Algeria. The nature of this resistance is of increasing concern with the emergence of new resistance determinants such as carbapenemases (classes A [e.g., KPC enzymes], B [VIM and NDM enzymes], and D [OXA enzymes]). In Algeria, the first carbapenemase described in Enterobacteriaceae was VIM-19 in 2010.⁸ Following that the KPC gene was reported in Enterobacteriaceae in 2010¹⁹ and KPC-3 in K. pneumoniae from a child's cerebrospinal fluid.¹⁵ Recently, Sassi et al. reported the detection of the bla_NDM-5 gene in E. coli.²⁰ Oxacillinase-type OXA-48 was first identified in Enterobacteriaceae in 2010.¹⁹ The OXA-48 enzyme was detected in an E. coli isolate from a patient from a province bordering Tunisia²¹ and recently in a K. pneumoniae isolate from patients in Algiers²² and Constantine²³ (Fig. 1 and Table 4). In our document, we describe the first detection of OXA-48-producing K. pneumoniae ST 404 and ST 219 in two cities (Annaba and Skikda) in northeastern Algeria. These results indicate the spread of the bla_OXA-48 gene to different regions in Algeria. K. pneumoniae ST 404 has been already reported in Korea²⁴ and ST 219 in Brazil.²⁵

FIG. 1.

Carbapenemase described in gram-negative bacilli clinical isolates from Algeria.

Table 4.

Carbapenemases Detected in Clinical Isolates Described in Different Cities in Algeria

Species	Enzyme described	Number of isolates	Year	Location	Reference
Enterobacteriaceae	KPC	1	2010	Unknown	Vaux et al.¹⁹
	OXA-48	1
E. coli	VIM-19	1	2010	Algiers	Robin et al.⁸
	NDM-5	3	2014	Annaba	Sassi et al.²⁰
	OXA-48	1	2014	Constantine	Agabou et al.²¹
K. pneumoniae	VIM-19	2	2010	Algiers	Robin et al.⁸
	OXA-48	1	2014	Algiers	Aggoune et al.²²
	KPC-3	1	2015	Sétif	Bakour et al.¹⁵
	OXA-48	1	2015	Constantine	Cuzon et al.²³
Providencia stuarti	VIM-19	2	2010	Algiers	Robin et al.⁸
P. aeruginosa	VIM-2	14	2013	Annaba	Touati et al.²⁷
	VIM-2	2	2014	Oran	Sefraoui et al.⁴
A. baumannii	OXA-58	16	2010	Tlemcen	Drissi et al.⁶
	OXA-23	1	2010	Unknown	Mugnier et al.⁷
	NDM-1	1
	OXA-94	1	2012	Oran	Boulanger et al.³⁶
	NDM-1	1	2012	Unknown	Bogaerts et al.³⁷
	OXA-23,	22
	OXA-58	1	2012	Annaba	Touati et al.³⁸
	OXA-23 and OXA-58	1
	OXA-23 and OXA-72	2	2012
	OXA-23	29		Sétif
	OXA-24	5
	OXA-23	2	2012	Tizi-Ouzou	Bakour et al.³⁹
	OXA-51	71	2012	Sétif and Tizi-Ouzou
	OXA-23	40
	OXA-24	3	2012	Tlemcen, Sétif, Sidi bel Abbes, oran, Tizi-Ouzou	Kempf et al.⁴⁰
	OXA-23 and OXA-24	3
	OXA-24,	17	2013	Tlemcen, Sidi bel
	OXA-23	40		Abbes, oran	Mesli et al.¹⁶
	NDM-1	5	2013	Oran
	OXA-51	47	2014	Sétif, Algiers, Bejaia
	OXA-23	33	2014	Algiers, Bejaia
	OXA-24	10	2014		Bakour et al.⁹
	NDM-1 and OXA-23	7	2014	Algiers
	NDM-1	4	2014	Sétif
	OXA-51	94	2015
	NDM-1	10
	OXA-24	19		Algiers	Khorsi et al.⁴¹
	OXA-23	63

In Algeria, few pieces of research have been conducted on the detection of OXA-48 enzyme. Routinely, in hospital laboratories, the use of imipenem antibiotic disks is more frequently used to detect OXA-48 than the other carbapenems such as ertapenem, but this enzyme confers high-level resistance to most β-lactam compounds, such as penicillins and cephalosporins, and variably affects carbapenems.²⁶ This was confirmed by our study, indeed, two OXA-48-producing K. pneumoniae, ST 404 and ST 219, which were resistant to ertapenem, were susceptible to imipenem.

For P. aeruginosa, only the bla_VIM-2 gene has been identified in Algeria by Touati et al. in Annaba²⁷ and Sefraoui et al. in Oran⁴ (Table 4 and Fig. 1). In this study, only two P. aeruginosa harbored the bla_VIM-4 gene (ALG_P19 from Annaba in a burns ward (GenBank accession no. KT698096) and the other ALG_P2 from Skikda in the pediatric ward (GenBank accession no. KT698097) (Table 3). The bla_VIM-4 gene was first described in P. aeruginosa from Thessaly (Larissa, Greece), and this finding was followed by an outbreak in this institution.^28,29 In another study, the bla_VIM-4 gene was reported by Libisch et al. in Hungary from seven hospitals³⁰ as it was also reported in Poland,³¹ France,³² Sweeden,³³ and Canada³⁴ (Fig. 2). Interestingly, a PubMed search failed to identify any published reports of coexpression of the bla_VIM-4 gene and OprD porin loss in identical clinical isolates of P. aeruginosa in North Africa. In this study, we report the first coexpression of the bla_VIM-4 gene and OprD in P. aeruginosa in two cities (Annaba and Skikda) in northeastern Algeria. These results indicate that the mutational inactivation of the OprD gene was the main mechanism for imipenem resistance in P. aeruginosa, as previously described.^5,4 Likewise, MLST revealed the presence of multiple clones (ST244, ST1076, ST 241, ST 227, and ST233), and the results of this work are consistent in five countries within the Mediterranean basin.³⁵ The ST1076 and ST244 clones were most frequently in keeping with the studies by Sefraoui et al. in Algeria⁴ and Al-bayssari et al. in Lebanon⁵ (Table 3).

FIG. 2.

Worldwide spread of VIM-4-producing Pseudomonas aeruginosa.

Similarly, in carbapenemase class B, NDM-1, the first carbapenemase, was identified in A. baumannii from Algerian patients transferred to France³⁶ and Belgium.³⁷ Following this, this enzyme was reported in western Algeria,¹⁶ in the Sétif and Algiers hospitals⁹ (Table 4 and Fig. 1). In the present study, two A. baumannii isolates produced the NDM-1 enzyme. This is the first result, showing the beginning of the spread of this enzyme in Annaba and Skikda. Of the oxacillinase enzymes, OXA-23 was the first enzyme to be found in A. baumannii.⁷ Following this, different groups of oxacillinases (OXA-24/72 and OXA-58) were described (Table 4 and Fig. 1). In the present study, five A. baumannii produced the OXA-23 enzyme, which has become endemic in Algeria. Finally, MLST analysis revealed the presence of two STs (2 and 85), which were found in A. baumannii from Algerian hospitals.¹¹ Our results suggest that these clones are now endemic within Algerian hospitals (Table 3).

In conclusion, our study reveals and confirms the dissemination of carbapenemase-producing gram-negative bacilli in Algerian hospitals and describes the first identification of VIM-4-producing P. aeruginosa ST 1076 and OXA-48-producing K. pneumoniae ST 404 and ST 219 in two cities (Annaba and Skikda) in northeastern Algeria. The emergence and spread of carbapenemase producers will limit the therapeutic options and threaten public health. Timely detection, implementation of infection control measures, formulation of an antibiotic policy, and preventive strategies to control dissemination of such strains are urgently required.

Data Access

The VIM-4 sequences of the two imipenem-resistant P. aeruginosa isolates have been deposited in GenBank under the accession numbers, KT698096 and KT698097.

Footnotes

Acknowledgments

The authors thank Linda Hadjadj for technical assistance and TradOnline for English corrections.

Funding Source

This work was partly funded by CNRS and IHU Méditerranée Infection.

Disclosure Statement

No competing financial interests exist.

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