Molecular Epidemiology and Drug Resistance Pattern of Carbapenem-Resistant Klebsiella pneumoniae Isolates from Iran

Abstract

The emergence and dissemination of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates and their involvement in several nosocomial outbreaks are of high concern. This study was conducted to investigate the genetic relatedness and molecular determinants of carbapenem resistance in 100 CRKP isolates. Susceptibility to carbapenems as well as other antibiotics was determined by using disk diffusion method. The Modified Hodge test was performed for detection of carbapenemase production. The minimum inhibitory concentrations of selected antibiotics were determined by broth microdilution method. The presence of bla_OXA-48, bla_KPC, bla_NDM, and bla_VIM carbapenemase genes was examined by PCR, and clonal relatedness of CRKP isolates was investigated by pulsed-field gel electrophoresis (PFGE) analysis. bla_OXA-48 was the most frequent carbapenemase gene (72%), followed by bla_NDM (31%). None of the isolates harbored bla_KPC and bla_VIM genes. PFGE separated the majority of isolates into 10 clusters, including the major clusters A and B, carrying bla_OXA-48, and clusters C and D, carrying bla_NDM, and 4 isolates had a unique PFGE pattern. An increased rate of colistin resistance (50%) was detected among the isolates. Tigecycline was found to be the most active agent against CRKP isolates. Our results revealed that high prevalence of bla_OXA-48 and bla_NDM carbapenamses and resistance to colistin are alarming threats, necessitating an immediate action to prevent the spread of carbapenem–colistin-resistant K. pneumoniae isolates in Iran.

Introduction

Infection caused by multidrug-resistant Gram-negative bacteria (MDR-GNB) poses a significant threat to infection control programs, and thus requires immediate action. Carbapenems are broad-spectrum β-lactam agents that often constitute the last line of defense against infections caused by MDR-GNB.¹ Enterobacteriaceae, which are resistant to carbapenems, known as carbapenem-resistant Enterobacteriaceae (CRE), have gradually emerged and brought us perilously close to none or very limited number of therapeutic options. Klebsiella pneumoniae is one of the most commonly isolated GNB in nosocomial infections and the most problematic member of Enterobacteriaceae, associated with the highest rates of carbapenem resistance.² Treatment of infections caused by MDR strains of K. pneumoniae are extremely challenging due to their resistance to most of antimicrobial drugs.

Reported possible therapeutic options for treatment of extensively drug-resistant (XDR) or pandrug-resistant (PDR) Enterobacteriaceae infections are commonly based on in vitro studies showing superiority of combination treatment over monotherapy regimens. The antimicrobial regimens mostly administrated in the combination therapy are fosfomycin, in combination with amikacin or colistin, colistin plus rifampin or tigecycline or carbapenem, and double-carbapenem regimen (a combination of ertapenem and doripenem).^3,4

Different classes of carbapenemases have been described in Enterobacteriaceae, including the Ambler class A β-lactamases (bla_KPC), class B metallo-β-lactamases (MBLs) (bla_VIM and bla_NDM), and class D β-lactamases (bla_OXA-48).⁵ Klebsiella pneumoniae carbapenemase (KPC) enzymes encoded by transferable plasmids are predominantly found in K. pneumoniae, and several reports have shown the rapid spread of KPC-producing K. pneumoniae isolates worldwide.^6–9 Infections caused by KPC-producing K. pneumoniae are associated with increased length of hospitalization and cost, high mortality rates, as well as frequent treatment failures.² Within the MBLs, VIM enzymes have been found to have a high affinity for carbapenems.¹⁰ The most recently recognized carbapenemase enzyme is the New Delhi metallo-β-lactamase (NDM), which has been found to be widespread among Enterobacteriaceae due to their plasmidic localization, which allows for rapid dissemination. NDM-1 is a broad-spectrum carbapenemase that confers resistance to all β-lactam antibiotics, except aztreonam. However, most NDM-1-producers also produce β-lactamases that can hydrolyze aztreonam, making these superbugs resistant to all β-lactams.² On the contrary, OXA-48 is one of the few members of Class D OXA β-lactamase family to possess notable carbapenem-hydrolyzing activity.¹¹ Following the first description of plasmid encoded OXA-48 from Turkey in 2004,¹² the bla_OXA-48 carbapenemase-producing Enterobacteriaceae have been reported worldwide, in particular, in Mediterranean countries, including Iran.¹³ Considering the global dissemination of CRE on a national and international scale and lack of data on molecular characteristics of carbapenem resistance in K. pneumoniae isolates from Iran, in the current study, we aimed at investigating the genetic relatedness and drug resistance profile of K. pneumoniae isolates from this geographic region.

Materials and Methods

Bacterial isolates

A total of 100 carbapenem-resistant isolates of K. pneumoniae were obtained from clinical samples of blood, wound, urine, sputum, and tracheal aspirates from October 2015 to September 2016. These isolates represented 10% of all isolates of K. pneumoniae (n = 992) cultured from patients hospitalized at Tehran Imam Khomeini hospital, a 1,400-bed teaching hospital, in Tehran during the study period. Wound samples were obtained by direct aspiration of pus or wound swabbing. For urine, growth of more than 100,000 CFU/mL was considered as positive culture. Direct smears from sputa were prepared, stained, and investigated by microscope.¹⁴ K. pneumoniae isolates were identified by microscopic examination and conventional biochemical methods and were confirmed by VITEK 2 system¹⁵.

Antimicrobial susceptibility testing

The susceptibility of bacterial isolates to various classes of antibiotics was determined by disk diffusion method (Kirby Bauer) with antibiotic discs (MAST Company, United Kingdom), according to the Clinical and Laboratory Standards Institute (CLSI) guideline¹⁶ using the following antibiotics: gentamicin (10 μg), amikacin (30 μg), piperacillin/tazobactam (100/10 μg), amoxicillin/clavulanic acid (20/10 μg), ampicillin (10 μg), chloramphenicol (30 μg), trimethoprim-sulphamethoxazole (1.25/23.75 μg), tetracycline (30 μg), tobramycin (10 μg), netilmicin (30 μg), cefepime (30 μg), cefoxitin (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), ceftriaxone (30 μg), cefuroxime (30 μg), ciprofloxacin (5 μg), aztreonam (30 μg), Ertapenem (10 μg), meropenem (10 μg), imipenem (10 μg), doripenem (10 μg), doxycycline (30 μg), minocycline (30 μg), tigecycline (15 μg), and colistin (10 μg). Food and Drug Administration interpretative criteria were used to test tigecycline susceptibility by disk diffusion method. The minimum inhibitory concentration (MIC) values of imipenem, meropenem, colistin, amikacin, cefotaxime, piperacillin–tazobactam, and ciprofloxacin were determined using broth microdilution method, according to the CLSI guidelines.¹⁶ For MIC determination of colistin a susceptible breakpoint of ≤2 mg/L and a resistant breakpoint of >2 g/mL was used according to guidelines described by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Escherichia coli ATCC 25922, E. coli ATCC 35218, and Pseudomonas aeruginosa ATCC 27853 were used as control organisms. MDR was defined as nonsusceptibility to ≥1 agents of at least three different antibiotic classes. XDR was defined as nonsusceptibility to at least one agent in all but two or fewer antimicrobial categories (e.g., bacterial isolates remain susceptible to only one or two antimicrobial categories). PDR was defined as nonsusceptibility to all agents in all antimicrobial categories.¹⁷ All isolates were tested for ESBL production using the phenotypic confirmatory test as instructed by CLSI.¹⁶ All the isolates with colistin MICs greater than 2 mg/L were screened for the presence of plasmid encoded colistin resistance gene mcr-1 using the previously described method.¹⁸ Also, isolates were examined for carbapenemase production using modified Hodge test (MHT), according to the CLSI guidelines.¹⁶ To evaluate MBL activity in Muller-Hinton agar plates, a combined disk test was performed as previously described by Nordmann et al.¹⁹ We used E. coli ATCC 25922 and K. pneumoniae ATCC 700603 for quality control. Isolates showing resistance to at least one of the carbapenems were stored in tryptic soy broth and glycerol at −70°C until use.

Detection of carbapenemase encoding genes

The genomic DNA was extracted from the bacterial strains by boiling the lysates prepared from the strains. In brief, a loopful of overnight culture was suspended in 200 μL of sterile TE buffer, boiled for 15 minutes at 100°C, and then centrifuged. A 1:10 diluted DNA in the supernatant fluid was used as a template for PCR.²⁰ The presence of bla_KPC, bla_VIM, bla_NDM, and bla_OXA-48 genes were examined by PCR using the previously described primers.²¹ The nucleotide sequences of bla_NDM were determined using the primers NDM-F-5′-GCCCAATATTATGCACCCGGTC and NDM-R-5′-AGCGCAGCTTGTCGGCCAT. K. pneumoniae AO 8053 (harboring bla_KPC), P. aeruginosa PO510 (harboring bla_VIM), E. coli MH01 (harboring bla_NDM), and K. pneumoniae Kp1514 (harboring bla_OXA-48) were used as positive controls.

Bacterial genotyping using pulsed-field gel electrophoresis

With some modifications, pulsed-field gel electrophoresis (PFGE) was performed according to the pulse net protocol from the Centers for Disease Control and Prevention (Atlanta).²² Chromosomal DNA, embedded into SeaKem Gold Agarose (Lonza, Rockland), was digested overnight with XbaI (Fermentas, Sylvius, Lithuania). Digested DNA fragments were separated after electrophoresis with CHEF-DR III System (Bio-Rad Laboratories) using the following program: initial pulse time for 2.2 seconds, final time for 54.2 seconds, and 120°C included angle at 6 V for 20 hours. Banding patterns were compared using GelJ software, Version 2.0. A dendrogram was constructed using the band-based Dice similarity coefficient and the unweighted pair group method with arithmetic mean. Banding patterns were interpreted using the criteria devised by Tenover et al.²³

Results

Bacterial isolates and antimicrobial susceptibility patterns

Most isolates were cultured from blood (31%), followed by tracheal aspirate (29%), urine (18%), and wound (11%). Isolates were all resistant to ampicillin, cefotaxime, ceftazidime, ceftriaxone, cefuroxime, and imipenem. The rates of resistance to other antibiotics, determined by disk diffusion method, were in the following order: gentamicin (93%), amikacin (50%), trimethoprim/sulfamethoxazole (98%), piperacillin/tazobactam (96%), aztreonam (98%), amoxicillin/clavulanic acid (98%), tobramycin (96%), netilmicin (94%), cefepime (99%), ciprofloxacin (92%), cefoxitin (98%), ertapenem (96%), meropenem (97%), doripenem (98%), and chloramphenicol (54%). Lower level of resistance to tetracycline (45%), doxycycline (44%), minocycline (31%), and tigecycline (9%) were observed. Moreover, 50 isolates (50%) showed resistance to “the last hope” anti-CRE antibiotic, colistin. None of the colistin-resistant isolates carried the plasmid encoded colistin resistance gene, mcr-1. According to our results, all isolates were classified as MDR, and 29% were XDR, but none was PDR. In the present work, we could not determine the different Klebsiella spp. species such as Klebsiella quasipneumoniae, Klebsiella variicola,²⁴ and Klebsiella quasivariicola²⁵ that could be found among our isolates identified biochemically as K. pneumoniae. In the case of K. variicola, a colistin-resistant isolate has been identified previously.²⁶ The MIC ranges obtained for all isolates (MIC90 and MIC50) and the percentage of resistant and susceptible isolates to each of the tested antibiotics are presented in Table 1.

Table 1.

Antimicrobial Susceptibility Profile of Carbapenem-Resistant Klebsiella pneumoniae Isolates

Antibiotic	S (%)	I (%)	R (%)	Range (mg/L)	MIC₉₀ (mg/L)	MIC₅₀ (mg/L)
Imipenem	0	0	100	16 → >256	>256	>256
Meropenem	2	1	97	0.03 → >256	>256	256
Colistin	50	—	50	0.06 → >256	256	2
Cefotaxime	0	0	100	16 → >1024	>1,024	1,024
Piperacillin/tazobactam	4	5	91	2 → >256	>256	256
Ciprofloxacin	4	4	92	0.2–>256	>256	64
Amikacin	46	6	48	0.2 → >256	>256	32

I, intermediate; R, resistant; S, susceptible.

Phenotypic and genotypic detection of carbapenemase production

A total of 9 isolates displayed a negative MHT result, while 91 were positive. Also, 41% and 55% of isolates were positive for MBL and ESBL production, respectively. PCR demonstrated that bla_OXA-48 and bla_NDM were detected in 72% and 31%, of isolates, respectively. None of the isolates was positive for bla_KPC and bla_VIM carbapenemase genes. A total of 18 isolates were found to coharbor bla_NDM-1 and bla_OXA-48 genes simultaneously. Also, 15 isolates were negative for all the 4 studied carbapenemase genes (Table 2).

Table 2.

Phenotypic and Genotypic Characteristics of the Carbapenem-Resistant Klebsiella pneumoniae Isolates from Iran

Isolate	Year	Ward	Source	IMI	MEM	CO (MIC (mg/L))	TGC	MBL	MHT	Carbapenemase	PFGE type
105	2016	Surgery	Trachea	R	R	R(256)	I	−	+	OXA-48		A1
98	2016	Internal medicine	Other	R	R	S(0.25)	I	−	+	OXA-48		A1
108	2015	ICU	Trachea	R	R	R(4)	I	−	+	OXA-48		A2
97	2016	Infectious diseases	Urine	R	R	R(64)	S	−	+	OXA-48		A2
85	2015	ICU	Blood	R	R	R(>256)	S	+	+	OXA-48		A2
81	2015	ICU	Wound	R	R	R(32)	S	−	+	OXA-48		A2
25	2016	ICU	Trachea	R	R	R(128)	I	+	+	OXA-48		A3
99	2016	Internal medicine	Other	R	R	R(128)	I	−	+	OXA-48		A3
79	2015	ICU	Trachea	R	R	R(>256)	I	−	−			A3
56	2015	SICU	Trachea	R	R	R(>256)	R	−	+	OXA-48		A3
59	2016	ICU	Trachea	R	R	S(0.06)	S	−	+	OXA-48		A4
40	2016	ICU	Blood	R	R	S(0.5)	S	+	+	OXA-48		A5
68	2015	ICU	Trachea	R	R	R(>256)	R	−	+	OXA-48		A6
21	2016	ICU	Urine	R	R	R(64)	I	−	+	OXA-48		A6
23	2016	ICU	Blood	R	R	R(32)	R	−	+	OXA-48		A7
15	2016	ICU	Trachea	R	R	S(0.06)	I	−	+	OXA-48		A7
46	2015	ICU	Trachea	R	R	S(0.5)	I	−	−			A8
74	2015	ICU	Trachea	R	R	R(>256)	I	−	+	OXA-48		A8
17	2016	Neurology	Trachea	R	R	S(0.25)	I	−	+	OXA-48		A8
16	2016	ICU	Trachea	R	R	R(64)	I	−	+	OXA-48		A8
12	2016	ICU	Trachea	R	R	R(64)	I	−	+	OXA-48		A8
9	2016	ICU	Trachea	R	R	R(64)	I	−	+	OXA-48		A8
8	2016	ICU	Trachea	R	R	R(64)	I	−	−			A8
69	2015	Orthopedics	Wound	R	R	R(8)	I	−	+	OXA-48		B1
65	2015	Neurology	Trachea	R	R	R(128)	I	−	+	OXA-48		B2
64	2015	Hematological	Wound	R	R	S(0.06)	I	−	+	OXA-48		B2
76	2015	Hematological	Blood	R	R	S(1)	S	−	+	OXA-48		B3
78	2015	Hematological	Blood	R	R	S(0.25)	S	−	+	OXA-48		B4
73	2015	ICU	Trachea	R	R	S(0.06)	I	−	+	OXA-48		B4
107	2016	ICU	Trachea	R	R	S(2)	I	−	+	OXA-48		B5
70	2015	ICU	Urine	R	R	R(128)	I	−	+	OXA-48		B5
57	2015	Internal medicine	Blood	R	R	S(0.06)	I	−	+	OXA-48		B6
52	2016	Hematological	Blood	R	R	S(0.06)	I	−	+	OXA-48		B7
51	2016	CCU	Blood	R	R	S(0.06)	S	−	+	OXA-48		B7
48	2015	Infectious diseases	Blood	R	R	S(0.5)	I	−	+	OXA-48		B8
37	2015	Internal medicine	Blood	R	R	S(0.5)	I	−	+	OXA-48		B8
34	2015	Liver transplant	Other	R	R	R(8)	S	+	+	OXA-48		B8
31	2015	Internal medicine	Blood	R	R	R(8)	S	−	+	OXA-48		B9
30	2015	Internal medicine	Blood	R	R	R(64)	I	−	+	OXA-48		B10
29	2015	infectious diseases	Urine	R	R	S(0.06)	S	−	+	OXA-48		B11
22	2016	Internal medicine	Other	R	R	R(16)	I	−	+	OXA-48		B12
3	2016	ICU	Trachea	R	R	R(128)	I	−	+	OXA-48		B13
62	2016	SICU	Trachea	R	R	S(0.25)	R	−	+	OXA-48		C1
50	2016	Internal medicine	Wound	R	R	S(0.5)	S	+	+	OXA-48	NDM-1	C2
44	2015	Internal medicine	Blood	R	R	S(0.5)	I	+	+		NDM-1	C3
47	2015	ICU	Blood	R	R	S(0.25)	I	+	+	OXA-48		C4
45	2015	Internal medicine	Other	R	R	S(1)	I	+	+		NDM-1	C5
41	2015	Internal medicine	Urine	R	R	S(0.25)	S	+	+		NDM-1	C5
53	2016	Internal medicine	Urine	R	R	R(256)	I	+	+	OXA-48	NDM-1	C6
6	2016	Emergency	Urine	R	R	S(0.25)	I	+	+		NDM-1	C7
26	2016	SICU	Trachea	R	R	R(16)	I	+	+	OXA-48	NDM-1	C8
27	2015	Internal medicine	Urine	R	R	S(2)	S	+	+		NDM-1	C9
20	2016	Internal medicine	Urine	R	R	S(0.5)	S	+	+	OXA-48	NDM-1	C10
91	2015	Internal medicine	Other	R	R	R(>256)	I	+	+	OXA-48	NDM-1	C11
54	2015	SICU	Blood	R	R	R(64)	S	+	+	OXA-48	NDM-1	C12
14	2016	Internal medicine	Sputum	R	R	S(0.06)	R	+	+			C13
13	2016	Emergency	Blood	R	R	S(0.25)	S	+	+	OXA-48	NDM-1	C13
4	2016	Internal medicine	Urine	R	R	S(0.25)	I	+	+		NDM-1	C14
121	2016	Emergency	Blood	R	R	S(1)	R	+	+		NDM-1	D1
117	2016	Emergency	Urine	R	R	R(256)	I	+	+		NDM-1	D2
120	2016	ICU	Blood	R	R	R(256)	S	+	+	OXA-48	NDM-1	D2
114	2016	ICU	Blood	R	R	R(256)	S	+	+	OXA-48	NDM-1	D2
118	2016	Urology	Trachea	R	R	R(128)	I	+	+	OXA-48	NDM-1	D2
119	2016	ICU	Wound	R	R	R(256)	I	+	+		NDM-1	D2
115	2016	Emergency	Blood	R	R	R(256)	S	+	+	OXA-48	NDM-1	D2
71	2015	Internal medicine	Urine	R	R	R(64)	I	+	+		NDM-1	D3
75	2016	ICU	Wound	R	R	R(4)	I	−	+	OXA-48		E1
42	2015	ICU	Blood	R	R	S(0.06)	I	+	+			E1
33	2015	Internal medicine	Wound	R	R	S(0.06)	R	−	+			E1
49	2016	Surgery	Blood	R	R	R(4)	S	−	+			E1
36	2015	CPR	Blood	R	R	S(0.06)	I	−	−			E1
35	2015	ICU	Blood	R	R	S(0.06)	I	−	+			E1
18	2016	ICU	Trachea	R	R	S(0.06)	R	−	−			E1
100	2016	Neurology	Trachea	R	R	R(8)	I	−	+	OXA-48		F1
106	2015	Infectious diseases	Other	R	R	R(16)	I	−	+	OXA-48		F1
84	2016	Surgery	Urine	R	R	R(256)	S	+	+		NDM-1	F2
82	2015	Internal medicine	Blood	R	R	S(2)	S	−	+	OXA-48		F2
89	2015	Liver transplant	Wound	R	R	S(2)	S	−	+	OXA-48		F2
88	2015	Infectious diseases	Trachea	R	R	R(64)	S	−	+	OXA-48		G1
101	2016	Internal medicine	Trachea	R	R	S(0.06)	S	+	+	OXA-48		G2
104	2016	Neurology	Wound	R	R	R(128)	S	+	+	OXA-48	NDM-1	G2
122	2016	CPR	Blood	R	R	R(128)	I	+	+	OXA-48	NDM-1	G2
39	2016	Internal medicine	Wound	R	R	S(0.5)	S	+	+	OXA-48	NDM-1	H1
11	2016	ICU	Trachea	R	R	S(1)	S	+	+	OXA-48	NDM-1	H2
5	2016	ICU	Trachea	R	R	S(0.5)	I	+	+	OXA-48	NDM-1	H2
109	2015	Orthopedics	Urine	R	R	S(1)	I	−	+	OXA-48		I1
110	2015	Orthopedics	Wound	R	R	S(2)	I	−	+	OXA-48		I1
60	2016	Internal medicine	Blood	R	R	R(64)	I	+	+	OXA-48		J1
43	2016	Infectious diseases	Blood	R	I	R(8)	S	−	+			J2
7	2016	Internal medicine	Trachea	R	S	S(0.25)	I	−	−			K
1	2016	NICU	Left eye	R	S	S(0.5)	I	−	−			L
61	2015	Nephrology	Urine	R	R	R(4)	I	−	−			M
87	2015	Internal medicine	Urine	R	R	S(2)	I	+	+		NDM-1	N
92	2015	Internal medicine	Left eye	R	R	R(64)	I	−	−			—
94	2015	Surgery	Other	R	R	R(256)	I	+	+	OXA-48	NDM-1	—
95	2015	Cancer	Blood	R	R	S(0.06)	S	+	+		NDM-1	—
102	2016	Emergency	Blood	R	R	S(2)	I	−	+	OXA-48		—
103	2016	Internal medicine	Urine	R	R	S(0.06)	I	+	+	OXA-48		—
111	2016	Infectious diseases	Urine	R	R	S(0.06)	R	−	+	OXA-48		—
123	2016	Neurology	Blood	R	R	R(4)	I	+	+	OXA-48	NDM-1	—

CO, colistin; IMI, imipenem; MEM, Meropenem; TGC, tigecycline; —, nontypable.

The clonal relatedness among the bacterial isolates

PFGE divided 89 isolates into 10 clusters (clusters A, B, C, D, E, F, G, H, I, and J consisted of 23, 19, 16, 8, 7, 5, 4, 3, 2, and 2 isolates, respectively), and 4 isolates were characterized with single unique PFGE pattern (types K, L, M, and N). PFGE could not yield DNA fingerprints for seven isolates, and therefore, these isolates could not be genotyped. The clusters A, B, and C were dominant clusters in this study. From 23 isolates classified in cluster A, 18 were from ICU ward, from which 13 strains were found to be isolated from tracheal aspirate. Also, 20 (86.9%) isolates in this cluster were found to harbor OXA-48, and 17 (73.9%) and 3 (13%) isolates were resistant to colistin and tigecycline, respectively. All isolates within cluster B (100%) were positive for OXA-48, from which 9 (47.3%) isolates were isolated from blood (Fig. 1, Table 2).

FIG. 1.

Pulsed-field gel electrophoresis pattern of XbaI-digested genomic DNA from carbapenem-resistant K. pneumoniae isolates. M, molecular marker (Lambda Ladder PFG Marker, Biolabs, New England), S, Salmonella H9812, Cluster A; No. 7, 8, 10 (A8) and 13 (A7), Cluster B; No. 2 (B13), Cluster C; No. 3 (C14), 5 (C7), 11 and 12 (C13), Cluster H; No. 4 and 9 (H2), PFGE type K; No. 6, PFGE type L; No. 1.

Discussion

Clinicians increasingly rely on carbapenem antibiotics to treat infections caused by MDR-GNB.²⁷ Among the carbapenem-resistant K. pneumoniae (CRKP) isolates studied here, we found high rates of resistance to β-lactams, aminoglycosides, and quinolones. Previously, we had found that 69.7% of K. pneumoniae isolates obtained from patients in Tehran were ESBL-producer, which were found to harbor TEM-1, SHV-5, SHV-11, SHV-12, and CTX-M-15 as the dominant ESBLs.²⁸ In the current study, we found that tetracyclines with resistance rate of <50% had higher activity against CRKP isolates compared with other classes of antibiotics, with the minocycline and tigecycline being the most active agents. About 9% of CRKP isolates were resistant to tigecycline. A SENTRY Antimicrobial Surveillance plan presented in 2016 reported that 2.6% of CRE from Latin America were tigecycline resistant.²⁹ Amikacin was found to be the most active agent among the aminoglycoside family of antibiotics. Despite being considered as a last line therapeutic option against CRKP, colistin was not as active as expected, with 50% of isolates being found to be resistant to this anti-CRE agent. Previous reports from Mexico, India, United Kingdom, and Italy revealed that prevalence of colistin resistance was 4%, 6%, 11%, and 36.1%, respectively.^5,30,31 Unfortunately, increased application of colistin therapy for infections caused by MDR-GNB has given rise to emergence of colistin-resistant GNB, moreover, several studies have reported outbreaks caused by colistin-resistant Enterobacteriaceae.^32–34 The prevalence of colistin-resistant K. pneumoniae is increasing worldwide, which is a serious threat to global health. As announced by Capone et al., diseases caused by colistin-resistant K. pneumoniae isolates have a high fatality rate in patients infected with these isolates.³¹ Currently, the emergence of transmissible colistin resistance is a global alert for human health and may speed up the progression of XDR-Enterobacteriaceae to PDR-Enterobacteriaceae and may also cause a universal expansion of pandrug resistance.³⁵ The results of this study revealed that bla_OXA-48 was the most frequently detected carbapenemase, followed by bla_NDM-1, and this finding is similar to that of other studies.^36,37 The bla_OXA-48 carbapenemase gene have been reported from France,³⁸ Spain,³⁹ Iran,¹³ Lebanon,³⁸ and Oman,⁴⁰ becoming one of the important resistance mechanisms in K. pneumoniae.^11,39 Due to the high prevalence of OXA-48 producers among Enterobacteriaceae in Turkey,⁴¹ as one of the countries bordering Iran, it may be concluded that migration, trade, or tourism between these two countries might contribute to the high prevalence of this gene among Iranian isolates.²⁰ Co-occurrence of bla_NDM-1 with bla_OXA-48 carbapenemase in E. coli and K. pneumoniae isolates has been reported in many studies.^42–44 Coexistence of the 2 bla_OXA-48 and bla_NDM-1 genes was observed in 18 CRKP isolates. The coexistence of different carbapenemase genes creates a challenge in the treatment of infections due to confined treatment options and the probability of global outbreak by cross-border travel.^45,46 Among the 91 CRE isolates with a positive MHT result, 85 were found to harbor the bla_NDM-1 and bla_OXA-48 alone or a combination of 2 genes. In the remaining six isolates, other carbapenemases that were not investigated might have been involved in carbapenem resistance. Genotypic methods are required to demonstrate clonal relatedness among K. pneumoniae isolates. Several different molecular typing methods have been used to study the epidemiology of infections caused by K. pneumoniae,⁴⁷ among which PFGE has been considered to be the gold standard method.⁴⁸ The PFGE analysis revealed a relatively high clonal diversity among the isolates. This diversity can be related to the variety of sources introduced to this center. Imam Khomeini hospital is the largest hospital in Iran, to which patients are referred from almost all parts of the country. Clusters B and A were the major clones recovered from different wards in this hospital. All isolates within clone B and 86.9% of isolates in clone A harbored bla_OXA-48 gene. The OXA-48, the most common carbapenemase in this study, was detected in all PFGE clusters except for the PFGE type K, indicating the clonal and nonclonal transfer of this gene. NDM-1 was detected in different PFGE clusters, including C, D, F, G, H, and PFGE type N with the exception of clusters A and B. These clones were presumably transferred to other wards by the hospital staff, patients, or equipment within the hospital. An epidemiological investigation is required to identify the origins of the dubious cross-transmission and the probability of a common source within the center or from an unknown environmental source.⁴⁹ Colistin resistance was observed in most clones, including clone D, A, B, and C, with prevalence of 87.5%, 73.9%, 42.1%, and 25%, respectively. Therefore, transmission of colistin-resistant strains cannot be attributed to a specific clone.

In summary, we reported an extensive survey for the emergence of CRKP isolates at the largest university hospital in Iran. According to antimicrobial susceptibility testing results, tigecycline was found to be the most active antibiotic against CRKP isolates. Most CRKP isolates harbored double carbapenemase genes, including bla_OXA-48 and bla_NDM-1. Increased rate of colistin resistance and high prevalence of bla_OXA-48 and bla_NDM-1 carbapenemases, which were found among the K. pneumoniae isolates in this study, pose an important public threat to infection control programs in Iran.

Footnotes

Acknowledgment

This study was funded by Tehran University of Medical Sciences (Project no: 30899).

Disclosure Statement

No competing financial interests exist.

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