Emergence of Multidrug-Resistant New Delhi Metallo-β-Lactamase-1-Producing Klebsiella pneumoniae in Egypt

Abstract

Despite expansion of the New Delhi metallo-β-lactamase-1 (NDM-1) worldwide, the incident of outbreaks regarding Egypt is still uncommon. In this survey, we denounce the emanation of multidrug-resistant NDM-1-producing Klebsiella pneumoniae in Egypt. We have reclaimed 46 unrepeatable carbapenem-resistant K. pneumoniae isolates at El-demerdash hospital, Ain Shams University, Cairo, Egypt. All the isolates showed a decreased sensitivity to imipenem and meropenem via the disc diffusion method. Among the isolates, 10 were proven as NDM-1 producers by utilizing the phenotypic methods (modified Hodge test and EDTA synergistic test) and specific PCR detection of NDM-1 encoding gene, bla_NDM-1. The isolates hosting the bla_NDM-1 showed an elevated resistance to several classes of β-lactam and non β-lactam antibiotics. All bla_NDM-1-harboring isolates have showed positivity for one or more other plasmid-mediated bla genes; in addition, the isolates carried class 1 integron. Enterobacterial repetitive intergenic consensus (ERIC)-PCR results revealed that majority of the isolates, including the NDM-1 producers, are unrelated to each other. This highlights the danger of horizontal transfer of plasmids encoding for such carbapenemases, including NDM-1, between the isolates of K. pneumoniae. In summary, this study has confirmed the incidence of bla_NDM-1 together with other bla genes among the K. pneumoniae isolates in Egypt. Control and prevention of infection can be achieved through early detection of resistance genes among bacterial isolates; through limiting the dispersal of these organisms.

Introduction

K lebsiella pneumoniae is a paramount human Gram-negative bacilli bacterial pathogen that is highly common in communities and hospitals. This pathogen can cause fatal infections in the respiratory tract, urinary tract, gastrointestinal tract, and the bloodstream.^1,2

Among used antibiotics, carbapenem is deemed the first therapeutic option of the critical infections brought about multi drug-resistant K. pneumoniae.^1,3,4 However, recently, there are increasing reports of carbapenem resistance through the isolates of K. pneumoniae in many countries as a consequence of extensive utilization of the carbapenems.^5,6 Carbapenem resistance in K. pneumoniae is mainly due to production of beta-lactamases that hydrolyze carbapenems (carbapenemases). It has been reported that three major classes of carbapenemases in K. pneumoniae are the source of nosocomial outbreaks including class A serine enzymes (the KPC type); class B metallo-β-lactamases (MBLs) or metal enzymes (the VIM and IMP types); and class D enzymes (the OXA-48 type).^1,7–9

Lately, an incoming MBL nominated New Delhi metallo-β-lactamase-1 (NDM-1) has been recognized from K. pneumoniae (strain 05-506), which was retrieved from a formerly hospitalized Swedish patient in India.¹⁰ NDM-1 has the ability to break down all the β-lactam antibiotics barring aztreonam, which is generally inactivated by other mechanisms including overproduction of the AmpC β-lactamase and/or extended-spectrum β-lactamases, and low protein expression of the outer membrane combined with an active efflux pump.^11,12 Moreover, the gene encoding for NDM-1, named bla_NDM-1, is located on a transmissible plasmid and its association with other resistant determinants leads to comprehensive drug resistance, which leaves only a few therapeutic choices.^10,13

Extensive surveys performed in many countries determined NDM-1-producing K. pneumoniae isolates.¹⁴ However, the existing data on propagation of the NDM-1-producing K. pneumoniae in Egypt is still limited. Therefore, this work strived to evaluate the prevalence of NDM-1-producing K. pneumoniae isolates among hospitals in Egypt. In addition, we investigated the co-production of other plasmid-mediated β-lactamases, including KPC, IMP, VIM, and OXA-48, among the isolates. Finally, the clonal alliance of the isolates was inspected by enterobacterial repetitive intergenic consensus (ERIC)-PCR.

Materials and Methods

Bacterial isolates

Forty-six unrepeatable carbapenem-resistant K. pneumoniae isolates were involved in the present survey. The isolates were regained from varied clinical samples at El-demerdash hospital, Ain Shams University, Cairo, Egypt between August 2013 and March 2015. The isolation, identification, and storage of the tested isolates were carried out according to the standard microbiological techniques.¹⁵ All the isolates were confirmed as K. pneumonia by PCR detection and sequencing of their 16s rRNA gene as outlined previously.¹⁶ Isolates were stockpiled in Tryptic soy broth containing 20% glycerol at −80°C. The Institutional Ethics Committee has established its consent for this study. Also, the participants have given their informed consent before the study.

Sensitivity testing

Antibiotic sensitivity testing of the isolates was fulfilled against different antibiotic groups including β-lactams (imipenem, meropenem, cefepime, and co-amoxiclav), aminoglycosides (gentamicin, amikacin, and tobramycin), quinolones (levofloxacin and ciprofloxacin), and colistin via the disc diffusion method as formerly outlined.^1,2 Moreover, the minimum inhibitory concentrations of the antibiotics were assessed by the broth dilution method as previously outlined.^1,2 The susceptibility to all antibiotics, except colistin, was elucidated with respect to the standards of Clinical and Laboratory Standards Institute (CLSI) 2013 guidelines.¹⁷ For colistin, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoint was used.¹⁸ In these experiments, quality control was established by utilizing the standard strain Escherichia coli ATCC 25922.

Phenotypic inspection of carbapenemases

The phenotypic inspection of the β-lactamases was accomplished by the modified Hodge test (MHT) according to CLSI.¹⁷ In this test β-lactamase production is considered positive when the test isolate allows the growth of a carbapenem-sensitive E. coli ATCC 25922 strain in the presence of a carbapenem disk (imipenem, 10 μg). In this test, positive and negative controls were established by using K. pneumoniae ATCC BAA-1705 and ATCC BAA-1706 strains, respectively.

In addition, the screening of metallo-β-lactamases was established as outlined previously,¹⁹ utilizing the EDTA synergistic test. In this test, two imipenem discs (10 μg), and one comprising anhydrous EDTA (292 μg) (Sigma Chemicals, St. Louis, MO), are placed 25 mm off each other on an inoculated Mueller–Hinton agar plate. By comparing the imipenem-EDTA disc to that of the imipenem disc alone, a rise in the inhibition zone diameter of >4 mm around the former, confirms positivity for metallo-β-lactamase production. In this test, quality control was established by utilizing the standard strain E. coli ATCC 25922.

Moreover, specific detection of KPC enzyme production was estimated by utilizing the boronic acid test as outlined previously.²⁰ In this test, antibiotic disc (imipenem, meropenem, or cefepime) and an inhibitor of KPC production (400 μg of benzene boronic acid; Sigma-Aldrich, Steinheim, Germany) are used. The tests were established by setting the antibiotics disks with or without boronic acid on an inoculated Mueller–Hinton agar plate. By comparing the antibiotic-boronic acid disk to that of the antibiotic disc alone, a rise in the diameter of the inhibition zone by ≥5 mm around the former, confirms positivity for KPC enzyme production. In this test, quality control was established by utilizing the standard strain E. coli ATCC 25922.

PCR detection and sequence analysis of the β-lactamases genes

Plasmid DNA of each isolate was subjected to PCR detection of the bla_NDM-1 gene by using the target specific primer set, NDM-Fm (5′-GGTTTGGCGATCTGGTTTTC-3′) and NDM-Rm (5′-CGGAATGGCTCATCACGATC-3′).²¹ The isolates were also examined for the presence of other plasmid-mediated β-lactamases genes including bla_KPC, bla_IMP, bla_VIM, and bla_OXA-48 using the primer sets, KPC-Fm (5′-CGTTGACGCCCAATCC-3′) and KPC-Rm (5′-ACCGCTGGCAGCTGG-3′); IMP-Fm (5′-CATGGTTTGGTGGTTCTTGT-3′) and IMP-Rm (5′-ATAATTTGGCGGACTTTGGC-3′); VIM-Fm (5′-ATTGGTCTATTTGACCGCGTC-3′) and VIM-Rm (5′-TGCTACTCAACGACTGAGCG-3′); and OXA-Fm (5′-ATGCGTGTATTAGCCTTATCGGC-3′) and OXA-Rm (5′-ACTTCTTTTGTGATGGCTTGGCGCA-3′), respectively.^22–24 In addition, genomic or plasmid DNA of each isolate was subjected to PCR detection of the integron structures (Int1, Int2, and Int3) by using the target-specific primer set, Int1-Fm (5′-ACGAGCGCAAGGTTTCGGT-3′) and Int1-Rm (5′-GAAAGGTCTGGTCATACATG-3′); Int2-Fm (5′-GTGCAACGCATTTTGCAGG-3′) and Int2-Rm (5′-CAACGGAGTCATGCAGATG-3′); Int3-Fm (5′-CATTTGTGTTGTGGACGGC-3′) and Int3-Rm (5′-GACAGATACGTGTTTGGCAA-3′), respectively as outlined previously.^1,2 In these experiments, PCR reactions without DNA templates were used as negative controls. Moreover, sequencing of purified PCR products of the genes were performed by Macrogen, Inc. (Seoul, South Korea). Sequence similarity search was done through NCBI internet-based BLAST search program.

Enterobacterial repetitive intergenic consensus-PCR

Genomic DNA of each isolate was subjected to ERIC-PCR using the primers ERIC-Fm (5′-ATGTAAGCTCCTGGGGATTAAC-3′) and ERIC-Rm (5′-AAGTAAGTGACTGGGGTGAGCG-3′)^25,26 Gel documentation system (Uvitec, Cambridge, United Kingdom) was used to analyze the resulted amplicons using Uvisoft software version 11.9. Schematic dendogram and homology clusters between isolates were designed by Uviband map according to the method of unweighted pair-group, which uses arithmetic averages algorithm (using UPGMA software). Isolates with >85% similarity were considered clonal.

Nucleotide sequence accession number

The full-length sequence of bla_NDM-1 gene of one of the positive isolates (43K), acquired from this work, was offered to DDBJ/EMBL/GenBank database and was given the accession number LC107774.

Statistical analysis

To evaluate the significance of difference in the results, each experiment was repeated at least three times, the data were represented as mean ± standard deviation, compared by Student's t-test, and the p-values less than 0.05 were considered significantly different. GraphPad Prism-version 5 (GraphPad Software, Inc., La Jolla, CA) was used to perform statistical analysis of the results.

Results

Clinical features and antibiotic sensitivity profiles of the isolates

Table 1 depicts the clinical features of the isolates. Clinical samples were compiled from patients hosting different pavilions of El-demerdash hospital including neuro intensive care unit (n = 13) 28%, surgical intensive care unit (n = 17) 37%, and burn intensive care unit (n = 16) 35%. The patients were suffering from various infections including respiratory tract infections (RTIs), urinary tract infections (UTIs), wound infections (WIs), and bacteremia. The isolates were recovered from the collected clinical samples including sputum (n = 21) 45.65% in case of RTIs, urine (n = 18) 39.13% in case of UTIs, pus (n = 4) 8.69% in case of WIs, and blood (n = 3) 6.52% in case of bacteremia. The samples were collected from both men and women in a ratio of 1:1.

Table 1.

Characteristics and Antibiotic Susceptibility Patterns of Klebsiella pneumoniae Isolates

					MIC (μg/ml) (Antibiotic susceptibility patterns)^a
Isolate No.	Sex	Ward	Specimen	Infection	IPM	MEM	FEP	AMC	CN	AK	TOB	LEV	CIP	CO
43K	F	NICU	Urine	UTI	32 (R)	16 (R)	256 (R)	512 (R)	256 (R)	128 (R)	64 (R)	0.5 (S)	0.5 (S)	0.25 (S)
50K	F	BICU	Pus	WI	16 (R)	32 (R)	256 (R)	256 (R)	256 (R)	64 (R)	1 (S)	0.5 (S)	0.5 (S)	0.25 (S)
68K	F	NICU	Sputum	RTI	32 (R)	16 (R)	256 (R)	256 (R)	128 (R)	128 (R)	128 (R)	0.5 (S)	0.5 (S)	0.5 (S)
102K	F	SICU	Sputum	RTI	32 (R)	16 (R)	256 (R)	512 (R)	256 (R)	256 (R)	256 (R)	1 (S)	1 (S)	0.25 (S)
107K	M	BICU	Pus	WI	32 (R)	16 (R)	512 (R)	512 (R)	1 (S)	1 (S)	1 (S)	32 (R)	32 (R)	0.25 (S)
109K	F	SICU	Sputum	RTI	32 (R)	16 (R)	512 (R)	512 (R)	256 (R)	128 (R)	1 (S)	32 (R)	64 (R)	0.25 (S)
204K	F	BICU	Urine	UTI	32 (R)	16 (R)	256 (R)	512 (R)	256 (R)	128 (R)	256 (R)	64 (R)	64 (R)	0.5 (S)
217K	F	SICU	Blood	Bacteremia	32 (R)	32 (R)	256 (R)	256 (R)	128 (R)	128 (R)	128 (R)	64 (R)	64 (R)	0.25 (S)
231K	F	BICU	Pus	WI	32 (R)	16 (R)	512 (R)	256 (R)	256 (R)	256 (R)	128 (R)	64 (R)	32 (R)	0.25 (S)
247K	F	SICU	Sputum	RTI	64 (R)	32 (R)	512 (R)	256 (R)	256 (R)	256 (R)	64 (R)	0.5 (S)	0.5 (S)	0.5 (S)
250K	F	SICU	Sputum	RTI	64 (R)	32 (R)	256 (R)	256 (R)	256 (R)	1 (S)	1 (S)	32 (R)	64 (R)	0.5 (S)
256K	M	BICU	Urine	UTI	64 (R)	32 (R)	256 (R)	256 (R)	128 (R)	128 (R)	128 (R)	64 (R)	32 (R)	0.25 (S)
257K	F	NICU	Urine	UTI	16 (R)	32 (R)	512 (R)	512 (R)	256 (R)	64 (R)	256 (R)	64 (R)	32 (R)	0.25 (S)
268K	F	SICU	Blood	Bacteremia	32 (R)	16 (R)	512 (R)	512 (R)	2 (S)	1 (S)	1 (S)	0.5 (S)	0.5 (S)	0.25 (S)
282K	F	BICU	Pus	WI	32 (R)	32 (R)	512 (R)	256 (R)	128 (R)	256 (R)	128 (R)	32 (R)	64 (R)	0.25 (S)
295K	M	SICU	Sputum	RTI	64 (R)	32 (R)	256 (R)	256 (R)	256 (R)	128 (R)	128 (R)	64 (R)	32 (R)	0.5 (S)
298K	M	SICU	Sputum	RTI	32 (R)	16 (R)	256 (R)	256 (R)	256 (R)	128 (R)	128 (R)	128 (R)	32 (R)	0.5 (S)
305K	F	NICU	Blood	Bacteremia	32 (R)	16 (R)	256 (R)	512 (R)	256 (R)	1 (S)	256 (R)	1 (S)	64 (R)	0.25 (S)
412K	M	NICU	Sputum	RTI	32 (R)	16 (R)	256 (R)	512 (R)	128 (R)	256 (R)	128 (R)	128 (R)	64 (R)	0.25 (S)
415K	M	BICU	Urine	UTI	32 (R)	16 (R)	256 (R)	256 (R)	256 (R)	256 (R)	256 (R)	1 (S)	32 (R)	0.25 (S)
418K	M	SICU	Sputum	RTI	32 (R)	16 (R)	256 (R)	256 (R)	128 (R)	256 (R)	64 (R)	64 (R)	64 (R)	0.25 (S)
425K	F	BICU	Urine	UTI	16 (R)	16 (R)	512 (R)	512 (R)	256 (R)	256 (R)	64 (R)	32 (R)	32 (R)	0.5 (S)
429K	M	NICU	Urine	UTI	16 (R)	32 (R)	512 (R)	256 (R)	256 (R)	256 (R)	64 (R)	64 (R)	32 (R)	0.5 (S)
435K	F	SICU	Urine	UTI	32 (R)	16 (R)	512 (R)	256 (R)	2 (S)	1 (S)	1 (S)	32 (R)	64 (R)	0.5 (S)
437K	M	SICU	Sputum	RTI	32 (R)	16 (R)	512 (R)	256 (R)	128 (R)	256 (R)	64 (R)	64 (R)	32 (R)	0.25 (S)
440K	M	BICU	Urine	UTI	64 (R)	32 (R)	512 (R)	256 (R)	128 (R)	128 (R)	64 (R)	64 (R)	32 (R)	0.25 (S)
441K	M	BICU	Urine	UTI	32 (R)	16 (R)	256 (R)	256 (R)	128 (R)	128 (R)	128 (R)	64 (R)	64 (R)	0.25 (S)
449K	F	NICU	Urine	UTI	32 (R)	16 (R)	256 (R)	256 (R)	128 (R)	64 (R)	128 (R)	32 (R)	32 (R)	0.5 (S)
464K	M	BICU	Sputum	RTI	32 (R)	16 (R)	256 (R)	256 (R)	256 (R)	128 (R)	128 (R)	64 (R)	64 (R)	0.25 (S)
467K	M	SICU	Urine	UTI	32 (R)	16 (R)	256 (R)	256 (R)	128 (R)	64 (R)	64 (R)	64 (R)	32 (R)	0.25 (S)
471K	M	NICU	Sputum	RTI	32 (R)	16 (R)	256 (R)	256 (R)	256 (R)	64 (R)	64 (R)	0.5 (S)	0.5 (S)	0.25 (S)
478K	F	BICU	Urine	UTI	32 (R)	16 (R)	512 (R)	256 (R)	128 (R)	64 (R)	64 (R)	64 (R)	64 (R)	0.25 (S)
482K	M	NICU	Urine	UTI	64 (R)	32 (R)	512 (R)	512 (R)	128 (R)	128 (R)	128 (R)	32 (R)	64 (R)	0.25 (S)
487K	M	NICU	Sputum	RTI	32 (R)	32 (R)	512 (R)	256 (R)	256 (R)	64 (R)	128 (R)	0.5 (S)	0.5 (S)	0.25 (S)
489K	F	SICU	Sputum	RTI	32 (R)	32 (R)	512 (R)	256 (R)	256 (R)	128 (R)	128 (R)	64 (R)	32 (R)	0.5 (S)
492K	M	NICU	Sputum	RTI	32 (R)	16 (R)	256 (R)	256 (R)	256 (R)	128 (R)	256 (R)	128 (R)	32 (R)	0.5 (S)
493K	M	BICU	Sputum	RTI	32 (R)	16 (R)	256 (R)	512 (R)	128 (R)	64 (R)	256 (R)	64 (R)	64 (R)	0.25 (S)
501K	M	SICU	Sputum	RTI	32 (R)	32 (R)	256 (R)	512 (R)	128 (R)	256 (R)	128 (R)	64 (R)	64 (R)	0.25 (S)
505K	F	SICU	Sputum	RTI	64 (R)	32 (R)	256 (R)	256 (R)	128 (R)	128 (R)	128 (R)	64 (R)	32 (R)	0.25 (S)
521K	M	BICU	Urine	UTI	32 (R)	16 (R)	512 (R)	256 (R)	128 (R)	1 (S)	1 (S)	32 (R)	64 (R)	0.25 (S)
525K	M	SICU	Sputum	RTI	32 (R)	16 (R)	256 (R)	512 (R)	256 (R)	128 (R)	256 (R)	64 (R)	32 (R)	0.5 (S)
539K	F	NICU	Urine	UTI	64 (R)	32 (R)	256 (R)	512 (R)	1 (S)	256 (R)	128 (R)	32 (R)	32 (R)	0.25 (S)
553K	M	SICU	Sputum	RTI	64 (R)	32 (R)	256 (R)	256 (R)	256 (R)	128 (R)	128 (R)	0.5 (S)	1 (S)	0.25 (S)
571K	F	BICU	Urine	UTI	32 (R)	16 (R)	256 (R)	256 (R)	128 (R)	1 (S)	256 (R)	1 (S)	32 (R)	0.25 (S)
575K	M	NICU	Sputum	RTI	16 (R)	16 (R)	512 (R)	256 (R)	128 (R)	64 (R)	64 (R)	32 (R)	32 (R)	0.5 (S)
589K	F	BICU	Urine	UTI	64 (R)	32 (R)	512 (R)	256 (R)	128 (R)	64 (R)	64 (R)	32 (R)	32 (R)	0.25 (S)

Interpretive breakpoints of antibiotic susceptibility are based on the CLSI criteria, except for colistin for which the EUCAST breakpoints are used.

AK, amikacin; AMC, Co-amoxiclav; CIP, ciprofloxacin; CN, gentamicin; CO, colistin; FEP, cefepime; IPM, imipenem; LEV, levofloxacin; MEM, meropenem; TOB, tobramycin.

BICU, burn intensive care unit; F, female; M, male; MIC, minimum inhibitory concentration; NICU, neuro intensive care unit; R, resistant; RTI, respiratory tract infection; S, sensitive; SICU, surgical intensive care unit; UTI, urinary tract infection; WI, wound infection.

The patterns of antibiotic sensitivity of the isolates are presented in Table 1. All isolates exhibited resistance to imipenem, meropenem, cefepime, and co-amoxiclav. Moreover, 42 isolates (91.30%) were gentamicin resistant, 39 (84.78%) were amikacin and tobramycin resistant, 34 (73.91%) were levofloxacin resistant and 37 (80.43%) were ciprofloxacin resistant. To the contrary, colistin displayed a strong efficacy versus the isolates with a susceptibility rate of 100%.

Determination of carbapenemase content

As shown in Table 2, 24 isolates (52.17%) showed positive results for MHT. In addition, 23 isolates (50%) showed positive results for the EDTA synergistic test. Moreover, 36 isolates (78.26%) showed positive results for boronic acid test.

Table 2.

Phenotypic and Genotypic Detection of β-Lactamases Among K. pneumoniae Isolates

				β-lactamases genes
Isolate No.	MHT	EDTA	Boronic acid test	bla _NDM-1	Other β-lactamases	Class I integrons
43K	−	+	+	Present	bla_KPC, bla_IMP	Present
50K	−	+	+		bla_KPC, bla_VIM	Present
68K	−	+	−		bla _VIM	Present
102K	+	−	+		bla _KPC
107K	+	+	−		bla _IMP	Present
109K	+	−	+		bla _KPC
204K	−	−	+		bla _KPC	Present
217K	−	−	+		bla _KPC
231K	+	+	−		bla _IMP	Present
247K	+	+	−		bla_VIM, bla_OXA-48	Present
250K	+	−	+		bla_KPC, bla_OXA-48	Present
256K	+	+	−		bla _VIM	Present
257K	+	−	+		bla_KPC, bla_OXA-48
268K	+	+	+	Present	bla_KPC, bla_VIM	Present
282K	+	−	+		bla_KPC, bla_OXA-48	Present
295K	+	+	−		bla _VIM	Present
298K	+	+	+		bla_KPC, bla_VIM	Present
305K	−	+	+		bla_KPC, bla_VIM	Present
412K	−	+	−	Present	bla_VIM, bla_OXA-48	Present
415K	−	−	+		bla _KPC
418K	−	−	+		bla _KPC	Present
425K	+	−	+		bla _KPC	Present
429K	−	−	+		bla _KPC
435K	+	+	−		bla _VIM	Present
437K	−	−	+		bla _KPC
440K	−	−	+		bla _KPC	Present
441K	−	+	+		bla_KPC, bla_VIM	Present
449K	+	+	+	Present	bla_KPC, bla_OXA-48	Present
464K	−	−	+		bla _KPC
467K	−	+	−		bla _VIM	Present
471K	−	−	+		bla _KPC
478K	−	−	+		bla _KPC	Present
482K	−	−	+		bla _KPC
487K	+	+	+	Present	bla_KPC, bla_VIM	Present
489K	−	−	+		bla _KPC
492K	+	−	+		bla _KPC
493K	+	+	+	Present	bla_KPC, bla_VIM	Present
501K	+	+	+	Present	bla_KPC, bla_IMP	Present
505K	+	+	+	Present	bla_KPC, bla_IMP	Present
521K	−	−	+		bla_KPC, bla_OXA-48	Present
525K	−	−	+		bla _KPC	Present
539K	+	−	+		bla _KPC
553K	+	+	+		bla_KPC, bla_IMP	Present
571K	+	+	−	Present	bla _OXA-48	Present
575K	+	+	+	Present	bla_KPC, bla_VIM	Present
589K	−	−	+		bla _KPC	Present

+, Positive; −, negative; EDTA, ethylenediaminetetraacetic acid synergistic test; MHT, modified Hodge test.

To confirm the phenotypic outcomes, PCR and sequencing were done to detect the genes encoding for the β-lactamases. As shown in Table 2, 10 isolates (21.73%) were found to be positive for bla_NDM-1. From the 10 bla_NDM-1-positive isolates, a majority (6 isolates, 60%) were recovered from sputum, while 3 isolates (30%) were recovered from urine, and only 1 isolate (10%) was recovered from blood.

Furthermore, other types of β-lactamases were inspected, including KPC, IMP, VIM, and OXA-48, where, 36 isolates (78.26%) were positive for bla_KPC, 6 isolates (13.04%) were positive for bla_IMP, 15 isolates (32.60%) were positive for bla_VIM, and 8 isolates (17.39%) were positive for bla_OXA-48. From the distribution of the resistance genes among the isolates, it is clear that all bla_NDM-1-harboring isolates carry other β-lactamases genes. The coexistence of bla_NDM-1 with bla_KPC and bla_VIM was established in 4 (8.69%) isolates, that of bla_KPC and bla_IMP was established in 3 (6.52%) isolates, that of bla_KPC and bla_OXA-48 was established in 1 (2.17%) isolate, that of bla_VIM and bla_OXA-48 was established in 1 (2.17%) isolate, and that of bla_OXA-48 alone was established in 1 (2.17%) isolate. Moreover, detection of the integron structures (Int1, Int2, and Int3) among the isolates revealed that 33 isolates (71.74%) carried class 1 integron (Int1). However, sequencing showed that all isolates harboring Int1 had no resistance gene cassettes.

Genetic alliance of the carbapenemase-producing K. pneumoniae isolates

As seen in Fig. 1, ERIC-PCR assessment revealed that the isolates are uncorrelated to each other, except for the isolates 471K and 482K, 505K and 525K, 501K and 553K, and 487K and 492K.

FIG. 1.

ERIC-PCR analysis of Klebsiella pneumoniae isolates. Gel documentation system Uvitec was used to analyze the resulted bands by Uvisoft software version 11.9. Schematic dendogram and homology clusters between isolates were designed by Uviband map based on the unweighted pair-group method using arithmetic averages algorithm (using UPGMA software). A genetic similarity index scale is shown in the left top side of the dendogram. ERIC, enterobacterial repetitive intergenic consensus.

Discussion

The increasing reports on the NDM-1-producing K. pneumoniae are a major concern worldwide. Even though NDM-1 is mostly endemic in India, other surveys have proposed that other countries may act as a pool of the NDM-1 producers.^1,2,4,14 In Egypt, Abdelaziz et al.²⁷ described the first flare-up of NDM-1-producing K. pneumonia, however, the data on the prevalence of NDM-1-producing K. pneumoniae is still limited. Here, we are reporting the presence of multidrug-resistant NDM-1-producing K. pneumoniae isolates in various clinical samples collected from El-demerdash hospital, Ain Shams University, Cairo, Egypt. In our hospital, NDM-1-producing K. pneumonia were equally derived from specimens collected from men and women hosting different wards including neuro intensive care unit, surgical intensive care unit, and burn intensive care unit.

According to the CLSI recommendations,¹⁷ the MHT is considered as a phenotypic verificatory test for the inspection of carbapenemase production in K. pneumoniae isolates with decreased inhibition zones for carbapenems in disc diffusion sensitivity testing. Yet, the sensitivity and specificity of this test for the inspection of carbapenemase production is a matter of debate.^1,28 In this study, although all the isolates are carbapenemases producers, 24 (52.17%) of the isolates presented positivity for the MHT, while, the rest of the isolates, 22 (47.82%) showed negative results. Different studies also reported misleading positive or negative results produced by the MHT in the detection of carbapenemase production among Enterobacteriaceae including K. pneumoniae.^1,28–30 To the contrary, the EDTA synergistic test that was done for the inspection of the metallo-β-lactamase production, including NDM-1, was found to be highly sensitive, where, all metallo-β-lactamase harboring K. pneumoniae (23 isolates) showed positive results. Moreover, boronic acid test was highly sensitive to detect KPC enzyme production, where, all KPC-possessing K. pneumoniae (36 isolates) showed positive results. Therefore, our data recommend the EDTA synergistic test and boronic acid test as simple and sensitive tools for inspection of the metallo-β-lactamase (including NDM-1) and KPC-producing K. pneumoniae isolates, respectively in any microbiology laboratory. These results are in agreement with other reports that highlighted the accuracy of the EDTA synergistic test and boronic acid test to detect the carbapenemases production among Enterobacteriaceae including K. pneumoniae.^1,20,28

Carbapenems are believed to be the most appropriate therapy of the critical infections that brought about multidrug-resistant Gram-negative bacteria. Unfortunately, as soon as carbapenem-resistant strains arise, treatment options are no longer available; except for limited examples such as aminglycosides, quinolones, colistin, and tigecycline.^1,3,4 Our data showed that the K. pneumoniae isolates, which possessed the bla_NDM-1 gene, were much resistant to different categories of β-lactam and non β-lactam antibiotics by disc diffusion sensitivity testing. All the isolates that possessed bla_NDM-1 revealed resistance to β-lactam antibiotics involving imipenem, meropenem, cefepime, and co-amoxiclav. On the other hand, all bla_NDM-1-positive isolates were sensitive to colistin, four were sensitive to levofloxacin, three were sensitive to ciprofloxacin, two were sensitive to amikacin, and one was sensitive to gentamicin and tobramycin. These results indicate that colistin is the ultimate therapeutic option for infections caused by multidrug-resistant K. pneumonia. Similar patterns of susceptibility to the non β-lactam antibiotics in the NDM-1-producing organisms have been described in other studies.^1,28,31,32

As a high level of resistance was observed to the different categories of the β-lactam antibiotics, we determined other important bla genes in the NDM-1-producing K. pneumoniae isolates using PCR. It was observed that all the isolates possessed one or two additional bla genes including bla_KPC, bla_IMP, bla_VIM, and bla_OXA-48. The frequency of bla_KPC, bla_IMP, bla_VIM, and bla_OXA-48 in the NDM-1-producing K. pneumoniae isolates were 80%, 30%, 50%, and 30% respectively. Former surveys also conveyed the presence of other bla genes in the NDM-1-producing K. pneumoniae isolates.^1,28,30 The incidence of other bla genes in all the bla_NDM-1-possessing isolates indicates the prevalence of several gene cassettes encoding for these carbapenemase among the isolates. Several gene cassettes encoding multiple carbapenemases have been described in previous studies.^1,33–35 In this study, each isolate possessing class 1 integron had no resistance gene cassettes, which indicates that class 1 integron was not the reason behind the prevalence of resistance genes.

ERIC-PCR is currently one of the most powerful tools used for the analysis of clonal alliance.^36–38 In our study, homology analysis (using ERIC-PCR) indicated that plasmid-mediated carbapenemases, including NDM-1, can be easily spread among the K. pneumoniae isolates. Although, the majority of the isolates were unrelated to each other, they carried two or more carbapenemases. These results suggest that the plasmids encoding for carbapenemases can be easily spread laterally among the K. pneumoniae isolates. These results are in agreement with former reports that clarified the horizontal transfer of plasmids encoding for carbapenemases among enterobacteriaceae including K. pneumoniae.^1,34,35

In conclusion, carbapenem resistance among K. pneumoniae has been considered as one of the most significant menaces to the global healthcare, and the prevalence of NDM-1-producing K. pneumoniae has heightened this threat. Here, we report the efflorescence of NDM-1-producing multidrug-resistant K. pneumoniae in Egypt. It has been observed that all the bla_NDM-1-positive isolates were hosting other plasmid-mediated bla genes. The dissemination of such plasmids between different clinically important bacterial species as K. pneumoniae, which accounts for one of the important bacterial pathogens in the hospital environments, may lead to serious public health issues. Therefore, the early detection of the bla_NDM-1-possessing K. pneumoniae isolates with any decreased sensitivity to the carbapenems is indispensable for the choice of the most appropriate antibiotic therapy and the application of efficient infection control measures. The appearance of such resistance patterns may be reduced by the restricted implementation of antimicrobials, especially for carbapenems and cephalosporins.

Footnotes

Acknowledgments

This study was performed at the Microbiology Department, Faculty of Pharmacy, Misr University for Science and Technology, 6th of October City, Egypt, in collaboration with the Microbiology and Immunology Department, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt.

Disclosure Statement

No competing financial interests exist.

References

Jin

, Shao

, Li

, Fan

, Bai

, and Wang

. 2015. Outbreak of multidrug resistant NDM-1 producing Klebsiella pneumonia from a Neonatal Unit in Shandong Province, China. PLoS One, 10:e0119571.

Wang

J.T.

, Wu

U.I.

, Lauderdale

T.L.

, Chen

M.C.

, Li

S.Y.

, Hsu

L.Y.

, and Chang

S.C.

. 2015. Carbapenem-nonsusceptible enterobacteriaceae in Taiwan. PLoS One, 10:e0121668.

Lee

C.M.

, Liao

C.H.

, Lee

W.S.

, Liu

Y.C.

, Mu

J.J.

, Lee

M.C.

, and Hsueh

P.R.

. 2012. Outbreak of Klebsiella pneumoniae carbapenemase-2 producing K. pneumoniae sequence type 11 in Taiwan in 2011. Antimicrob. Agents Chemother., 56:5016–5022.

Poirel

, Lascols

, Bernabeu

, and Nordmann

. 2012. NDM-1 producing Klebsiella pneumoniae in Mauritius. Antimicrob. Agents Chemother., 56:598–599.

Nordmann

, Naas

, and Poirel

. 2011. Global spread of carbapenemase producing Enterobacteriaceae. Emerg. Infect. Dis., 17:1791–1798.

Tseng

S.H.

, Lee

C.M.

, Lin

T.Y.

, Chang

S.C.

, and Chang

F.Y.

. 2011. Emergence and spread of multi-drug resistant organisms: think globally and act locally. J. Microbiol. Immunol. Infect., 44:157–165.

Peirano

, Seki

L.M.

, Val Passos

V.L.

, Pinto

M.C.

, Guerra

L.R.

, and Asensi

M.D.

. 2008. Carbapenem-hydrolysing beta-lactamase KPC-2 in Klebsiella pneumoniae isolated in Rio de Janeiro, Brazil. J. Antimicrob. Chemother., 63:265–268.

Poirel

, Revathi

, Bernabeu

, and Nordmann

. 2011. Detection of NDM-1-producing Klebsiella pneumoniae in Kenya. Antimicrob. Agents Chemother., 55:934–936.

, Wei

, Ji

, Du

, Shen

, and Yu

. 2011. ST11, the dominant clone of KPC-producing Klebsiella pneumoniae in China. J. Antimicrob. Chemother., 66:307–312.

10.

Yong

, Toleman

M.A.

, Giske

C.G.

, Cho

H.S.

, Sundman

, Lee

, and Walsh

T.R.

. 2009. Characterization of a new metallo-beta-lactamase gene, bla (NDM-1), and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence 14 from India. Antimicrob. Agents Chemother., 53:5046–5054.

11.

Dallenne

, Da Costa

, Decré

, Favier

, and Arlet

. 2010. Development of a set of multiplex PCR assays for the detection of genes encoding important beta-lactamases in Enterobacteriaceae. J. Antimicrob. Chemother., 65:490–495.

12.

Yang

F.C.

, Yan

J.J.

, Hung

K.H.

, and Wu

J.J.

. 2012. Characterization of ertapenem resistant Enterobacter cloacae in a Taiwanese university hospital. J. Clin. Microbiol., 50:223–226.

13.

Marra

2011. NDM-1: a local clone emerges with worldwide aspirations. Future Microbiol., 6:137–141.

14.

Berrazeg

, Diene

, Medjahed

, Parola

, Drissi

, Raoult

, and Rolain

. 2014. New Delhi metallo-beta-lactamase around the world: an eReview using Google Maps. Euro Surveill., 19:20809.

15.

Collee

J.G.

, Miles

R.S.

, and Wan

. 1996. Tests for the identification of bacteria. In Collee

J.G.

, Fraser

A.G.

, Marmion

B.P.

, and Simmons

(eds.), Mackie and McCartney Practical Medical Microbiology, 14th ed. Churchill Livingstone, Edinburgh, pp. 131–150.

16.

Elgaml

, Hassan

, Barwa

, Shokralla

, and El-Naggar

. 2013. Analysis of 16S ribosomal RNA gene segments for the diagnosis of Gram negative pathogenic bacteria isolated from urinary tract infections. Afr. J. Microbiol. Res., 7:2862–2869.

17.

Clinical and Laboratory Standards Institute (CLSI). 2013. Performance Standards for Antimicrobial Susceptibility Testing Twentieth Informational Supplement, M100-S23. CLSI, Wayne, PA.

18.

European Committee on Antimicrobial Susceptibilities Testing (EUCAST). 2014. Breakpoint Tables for Interpretation of MICs and Zone Diameters. EUCAST. Available at www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/Breakpoint_table_v_4.0.pdf (Accessed September 9, 2014).

19.

Goldstein

, Lee

M.D.

, Sanchez

, Hudson

, Phillips

, Register

, Grady

, Liebert

, Summers

A.O.

, White

D.G.

, and Maurer

J.J.

. 2001. Incidence of class 1 and 2 integrases in clinical and commensal bacteria from livestock, companion animals, and exotics. Antimicrob. Agents Chemother., 45:723–726.

20.

Tsakris

, Themeli-Digalaki

, Poulou

, Vrioni

, Voulgari

, Koumaki

, Agodi

, Pournaras

, and Sofianou

. 2011. Comparative evaluation of combined-disk tests using different boronic acid compounds for detection of Klebsiella pneumonia carbapenemase-producing enterobacteriaceae clinical isolates. J. Clin. Microbiol., 49:2804–2809.

21.

Nordmann

, Poirel

, Carrër

, Toleman

M.A.

, and Walsh

T.R.

. 2011. How to detect NDM-1 producers. J. Clin. Microbiol., 49:718–721.

22.

Jeon

B.C.

, Jeong

S.H.

, Bae

I.K.

, Kwon

S.B.

, Lee

, Young

, Lee

J.H.

, Song

J.S.

, and Lee

S.H.

. 2005. Investigation of a nosocomial outbreak of imipenem-resistant Acinetobacter baumannii producing the OXA-23 beta-lactamase in korea. J. Clin. Microbiol., 43:2241–2245.

23.

Teo

, Ngan

, Balm

, Jureen

, Krishnan

, and Lin

. 2012. Molecular characterization of NDM-1 producing Enterobacteriaceae isolates in Singapore hospitals. Western Pac. Surveill. Response J., 3:19–24.

24.

Venkatachalam

, Teo

, Balm

M.N.

, Fisher

D.A.

, Jureen

, and Lin

R.T.

. 2012. Klebsiella pneumoniae carbapenemase-producing enterobacteria in hospital, Singapore. Emerg. Infect. Dis., 18:1381–1383.

25.

Duan

, Chai

, Liu

, Zhang

, Qi

, Gao

, Wang

, Cai

, Miao

, Yao

, and Schlenker

. 2009. Source identification of airborne Escherichia coli of swine house surroundings using ERIC-PCR and REP-PCR. Environ. Res., 109:511–517.

26.

Versalovic

, Koeuth

, and Lupski

J.R.

. 1991. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res., 19:6823–6831.

27.

Abdelaziz

M.O.

, Bonura

, Aleo

, Fasciana

, and Mammina

. 2013. NDM-1- and OXA-163-producing Klebsiella pneumoniae isolates in Cairo, Egypt, 2012 J. Glob. Antimicrob. Resist., 1:213–215.

28.

Bora

, and Ahmed

. 2012. Detection of NDM-1 in clinical isolates of Klebsiella pneumoniae from northeast India. J. Clin. Diagn. Res., 6:794–800.

29.

Carvalhaes

C.G.

, Picao

R.C.

, Nicoletti

A.G.

, Xavier

D.E.

, and Gales

A.C.

. 2010. The cloverleaf test (modified Hodge test) for detecting carbapenemase production in Klebsiella pneumoniae: be aware of false positive results. J. Antimicrob. Chemother., 65:249–251.

30.

Wang

, Chen

, Guo

, Xiong

, Hu

, Zhu

, and Zhang

. 2011. The occurrence of false positive results for the detection of carbapenemases in the carbapenemase-negative Escherichia coli and the Klebsiella pneumoniae isolates. PLoS One, 6:e26356.

31.

Bharadwaj

, Joshi

, Dohe

, Gaikwad

, Kulkarni

, and Shouche

. 2012. Prevalence of New Delhi metallo-β-lactamase (NDM-1)-positive bacteria in a tertiary care centre in Pune, India. Int. J. Antimicrob. Agents., 39:265–266.

32.

Seema

, Ranjan Sen

, Upadhyay

, and Bhattacharjee

. 2011. Dissemination of the New Delhi metallo-β-lactamase-1 (NDM-1) among Enterobacteriaceae in a tertiary referral hospital in north India. J. Antimicrob. Chemother., 66:1646–1647.

33.

Khan

A.U.

, and Nordmann

. 2012. NDM-1-producing Enterobacter cloacae and Klebsiella pneumoniae in diabetic foot ulcers in India. J. Med. Microbiol., 61:454–456.

34.

Dortet

, Poirel

, and Nordmann

. 2014. Worldwide dissemination of the NDM-Type carbapenemases in gram-negative bacteria. Biomed. Res. Int., 2014:249856.

35.

Fluit

A.C.

, and Schmitz

F.J.

. 1999. Class 1 integrons, gene cassettes, mobility, and epidemiology. Eur. J. Clin. Microbiol. Infect. Dis., 18:761–770.

36.

Fouad

, Attia

A.S.

, Tawakkol

W.M.

, and Hashem

A.M.

. 2013. Emergence of carbapenem-resistant Acinetobacter baumannii harboring the OXA-23 carbapenemase in intensive care units of Egyptian hospitals. Int. J. Infect. Dis., 17:e1252–e1254.

37.

Liu

P.Y.

, and Wu

W.L.

. 1997. Use of different PCR-based DNA fingerprinting techniques and pulsed-field gel electrophoresis to investigate the epidemiology of Acinetobacter calcoaceticus–Acinetobacter baumannii complex. Diagn. Microbiol. Infect. Dis., 29:19–28.

38.

Medina-Presentado

J.C.

, Seija

, Vignoli

, Pontet

, Robino

, Cordeiro

N.F.

, Bado

, García-Fulgueiras

, Berro

, Bazet

, Savio

, and Rieppi

. 2013. Polyclonal endemicity of Acinetobacter baumannii in ventilated patients in an intensive care unit in Uruguay. Int. J. Infect. Dis., 17:e422–e427.