New Delhi metallo-β-lactamase and OXA-48 carbapenemases in Gram-negative bacilli isolates in Libya.

To the Editor

Carbapenems (i.e. ertapenem, imipenem, meropenem, and doripenem) are second line antimicrobials reserved to treat serious infections, including those caused by Gram-negative bacilli (GNB) producing extended-spectrum β-lactamses (ESBLs). ESBLs mediate resistance among GNB to β-lactam drugs, including third generation cephalosporins. Emergence of carbapenemases-producing GNB (mainly Acinetobacter baumanii, Pseudomonas aeruginosa, and members of the family Enterobacteriaceae) in the last decade is a serious health problem globally. Carbapenemases are members of three molecular groups of β-lactamases, Ambler class A, B (i.e. metallo β-lactamases [MBLs]), and D (i.e. oxacillinases) (1, 2). Recently, the novel carbapenemase New Delhi MBL (NDM) was reported for the first time in Tunisia in Klebsiella pneumoniae isolated from a female patient transferred from Libya (3). However, there is little information regarding genes associated with carbapenem resistance among GNB in Libya.

Included in the study were eight GNB isolates that are resistant to at least one carbapenem (i.e. ertapenem) obtained in 2013 and 2014 from different clinical specimens from patients at the time an infection occurred. Patients were aged between 10 and 87 years (mean=37.1 years). Information about patients and the organisms isolated from them included in the study are shown in Table 1. In this prospective investigation, clinical specimens were collected under approved ethical standards, and the study was reviewed and approved by the Academy of Graduate Studies, Tripoli, Libya.

Table 1

Information about patients and their isolated organisms

Patient	Gender	Age (years)	Hospital/department	Hospital admission	Specimen	Organism isolated
1	Male	30	TMC/ICU	Inpatient	Endotracheal tube tip	Acinetobacter baumannii
2	Female	57	TMC/GSICU	Inpatient	Suction tip	Acinetobacter baumannii
3	Female	46	TMC/ICU	Inpatient	Sputum	Pseudomonas aeruginosa
4	Male	25	BPSH/ICU	Inpatient	Wound swab	Pseudomonas putida
5	Male	10	BPSH	Outpatient	Wound swab	Escherichia coli
6	Male	87	TMC	Outpatient	Ear swab	Klebsiella pneumoniae
7	Female	42	TMC/Endo	Inpatient	Bedsore swab	Klebsiella pneumoniae
8	Male	30	TMC/GSICU	Inpatient	Wound swab	Enterobacter gergoviae

TMC=Tripoli Medical Center, BPSH=Burn and Pediatric Surgery Hospital, ICU=intensive care unit, GSICU=general surgical ICU, OP=outpatient, Endo=endocrinology.

TMC and BPSH are both located in Tripoli.

All specimens were cultured on plates of blood agar and MacConkey agar (Oxoid Ltd., Basingstoke, Hampshire, UK) and incubated at 37°C for 24–48 h. Isolated organisms were identified to the species level and tested for their susceptibility to a variety of antimicrobial agents by the BD Phoenix Automated Microbiology System (PAMS, MSBD Biosciences, Sparks, MD, USA) as recommended by the manufacturer. PAMS provides antimicrobial susceptibility results as susceptible (S), intermediate susceptible (I), or resistant (R) and are interpreted according to Clinical and Laboratory Standards Institute criteria (4). Quality control strains used included Escherichia coli ATCC 25922, K. pneumoniae ATCC 700603, and P. aeruginosa ATCC 27853 (ATCC, LGC Standards S.r.l., Italy). Furthermore, six of the GNB isolates were tested phenotypically for the production of MBLs by the MIC Test Strip MBL (Liofilchem, Rosetodegli Abruzzi, Italy) as recommended by the manufacturer.

All eight isolates were examined for genes encoding ESBLs blaCTX-M, blaGES, blaVEB, blaPER, or blaBES and carbapenemases blaKPC, blaVIM, blaNDM, or blaOXA-48 by polymerase chain reaction (PCR) using primers and conditions described previously (5, 6). In addition, both A. baumanii isolates were further tested for carbapenemase genes blaOXA-23-like, blaOXA-24/40-like, blaOXA-143-like, blaOXA-51-like, and blaOXA-58-like using PCR as above.

All A. baumanii and Pseudomonas sp. isolates were resistant to the three carbapenems as well as to nearly all other antimicrobials that were used. Only colisitin showed excellent activity against the GNB isolates investigated (100% susceptible) followed by amikacin (50% susceptible). Furthermore, all organisms were multidrug resistant (MDR; resistance to three or more antimicrobial groups). Table 2 shows the antimicrobial susceptibility of the investigated GNB isolates.

Table 2

Antimicrobial susceptibility and metallo-β-lactamases of Gram-negative bacilli isolates from different clinical specimens in Tripoli, Libya

		Antimicrobial agent
No.	Organism	AM	CN	EP	IP	MP	CX	CR	CT	CZ	CP	AN	PT	TS	CF	LF	CL	MBLs
1	Acinetobacter baumannii	R	R	R	R	R	R	R	R	R	R	R	R	R	R	R	S	+
2	Acinetobacter baumannii	R	R	R	R	R	R	R	R	R	R	R	R	S	R	R	S	ND
3	Pseudomonas aeruginosa	R	R	R	R	R	R	R	R	I	I	I	S	R	R	R	S	+
4	Pseudomonas putida	R	R	R	R	R	R	R	R	R	R	R	R	R	S	S	S	+
5	Escherichia coli	S	R	R	S	S	R	R	R	S	R	R	S	R	R	R	S	−
6	Klebsiella pneumoniae	S	S	R	R	R	R	R	R	R	R	R	R	R	R	R	S	+
7	Klebsiella pneumoniae	S	R	R	I	R	R	R	R	R	R	R	R	S	R	R	S	−
8	Enterobacter gergoviae	S	R	R	S	R	R	R	R	R	R	R	R	S	R	R	S	ND

AK=amikacin, CN=gentamicin, EP=ertapenem, IP=imipenem, MP=meropenem, CX=cefoxitin, CR=cefuroxime, CT=ceftriaxone, CZ=ceftazidime, CP=cefepime, AN=aztronam, PT=pipracillin-tazobactam, TS=trimethoprim-sulfamethoxazole, CF=ciprofloxacin, LF=levofloxacin, CL=colisitin. S=susceptible, I=intermediate susceptible, R=resistant, MTLs=metallo β-lactamases tested phenotypically, += positive, −=negative, ND=not done.

Of the ESBLs genes examined, blaCTX-M was detected in one K. pneumoniae and in the Enterobacter gergoviae isolate, and blaGES in the P. aeruginosa isolate. Other ESBL genes examined were not detected in all eight GNB isolates. Of the carbapenemase genes investigated, blaNDM was detected in one K. pneumoniae, and blaOXA-48 in E. gergoviae and in another K. pneumoniae isolate. On the other hand, the carbapenemase gene blaKPC was not detected in all isolates investigated. Table 3 shows genes encoding ESBLs and carbapenemases in the GNB isolates from Tripoli, Libya.

Table 3

Genes encoding extended-spectrum β-lactamases (ESBLs) and carbapenemases in Gram-negative bacilli from Tripoli, Libya

		ESBLs genesa		Carbapenemase genesa,b
No.	Organism	blaCTX-M	bla GES	bla NDM	bla VIM	bla OXA-48	bla OXA-23-like	bla OXA-51-like
1	Acinetobacter baumannii	N	N	N	N	N	P	P
2	Acinetobacter baumannii	N	N	N	N	N	P	P
3	Pseudomonas aeruginosa	N	P	N	N	N	ND	ND
4	Pseudomonas putida	N	N	N	P	N	ND	ND
5	Escherichia coli	N	N	N	N	N	ND	ND
6	Klebsiella pneumoniae	P	N	P	N	N	ND	ND
7	Klebsiella pneumoniae	N	N	N	N	P	ND	ND
8	Enterobacter gergoviae	P	N	N	N	P	ND	ND

ESBLs genes blaVEB, blaPER and blaBES and carbapenemase gene blaKPC were not detected in all eight isolates tested.

Carbapenemases genes blaOXA-24/40-like, blaOXA-58-like and blaOXA-143 were not detected in both A. baumanii isolates examined.

N=negative, P=positive, ND=not done.

Oxacillinases are mainly present in Acinetobacter sp. (7), and some of them are intrinsic to the organism (e.g. OXA-51) while others are acquired (e.g. OXA-23). bla OXA-23-like and bla OXA-51-like were found in both A. baumanii isolates. Recently, Mathlouthi et al. (8) reported MDR A. baumannii isolates harboring bla OXA-23-like, bla OXA-24-like, and bla OXA-48-like genes in Libya.

On the other hand, we identified OXA-48 in one K. pneumoniae as well as in the E. gergoviae isolate. Kocsis et al. (9) reported K. pneumoniae harboring OXA-48 carbapenemase in a Libyan refugee in Italy. In addition, several studies reported OXA-48 carbapenemase-producing K. pneumoniae in patients transferred from Libya to neighboring and European countries (e.g. Tunisia, Denmark, and Slovenia) (3, 10–12). However, there are no reports of bla OXA-48-positive K. pneumonia and E. gergoviae from Libya.

Both bla CTX-M and bla NDM were detected in Klebsiella pneumoniae isolated from an ear swab from a female outpatient with an ear infection. This isolate was also positive for MBLs by the phenotypic method used (Table 2). K. pneumoniae harboring bla NDM has not been reported from Libya in the past.

Genes coding for the ESBLs and carbapenemases tested were not detected in the E. coli isolate. However, the organism was ESBL-positive phenotypically (data not shown), which may suggest that other ESBLs not investigated in this work might be responsible for its resistance to the third generation cephalosporin.

In conclusion, to the authors’ knowledge this is the first report from Libya of K. pneumoniae harboring bla NDM and bla CTX-M, K. pneumoniae harboring bla OXA-48, and E. gergoviae harboring bla OXA-48 and bla CTX-M. Emergence of MDR GNB harboring genes coding for carbapenemases will undoubtedly limit the use of carbapenems in treating serious infectious in the country and also in the nearby countries.