Detection of Colistin Resistance Gene mcr-1 in Hypervirulent Klebsiella pneumoniae and Escherichia coli Isolates from an Infant with Diarrhea in China

LETTER

In recent years, antimicrobial resistance has increasingly been recognized as a significant problem that transcends international boundaries. With the global increase in the prevalence of carbapenem-resistant Enterobacteriaceae, polymyxins have been resurrected as a last-resort treatment option (1). Thus, the first decade of the 21st century witnessed a dramatic increase in the consumption of colistin (2). Heavy use of colistin in agriculture has been reported in China and other countries, and, inevitably, colistin resistance has been increasing (3). Resistance to polymyxins mainly depends on modification of lipopolysaccharide (LPS), which is often chromosomally mediated (4). Recently, Liu et al. identified the plasmid-mediated colistin resistance gene mcr-1 in animals and humans in China (5), which was followed by reports from several other countries (6–8). In this report, we describe the detection of mcr-1 in Klebsiella pneumoniae (belonging to capsular serotype K1) and Escherichia coli isolates from a stool specimen of an infant in China.

The patient displayed diarrhea of undetermined origin for 7 days and cough and fever for 5 days. He was transferred to a hospital in Hangzhou, China, in December 2015 after therapy at a local hospital failed to alleviate symptoms. After exclusion of dysentery and intussusception, the patient was diagnosed with acute enteritis and acute bronchitis. Tests for rotavirus, adenovirus, Campylobacter jejuni, and respiratory viruses were all negative. On the second day following admission, stool samples were collected and submitted for etiological diagnosis. K. pneumoniae 15451-1 and E. coli 15451-2 were isolated simultaneously. Following treatment with piperacillin-tazobactam (100 mg given every 8 h [q8h]) for 7 days, the cough had been alleviated but the diarrhea continued. The therapeutic regimen was changed to imipenem (50 mg q6h), and the patient improved and was discharged after 10 days of hospitalization.

Isolates were identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), and antimicrobial susceptibility profiles were determined using the agar dilution method recommended by the Clinical and Laboratory Standards Institute (CLSI) (9). The MIC of colistin was determined according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints (10). Both of the isolates presented polymyxin-resistant phenotypes (16 μg/ml for 15451-1 and 8 μg/ml for 15451-2) and were confirmed to be positive for mcr-1 while negative for resistant mutations in pmrAB, PhoPQ, or mgrB by PCR and sequencing using previously reported primers (5). In addition, strain 15451-1 was positive for AmpC-type beta-lactamase gene bla_DHA-1 and demonstrated a hypermucoviscous phenotype, as confirmed by a string test (11). Further detection of the K1 capsular serotype indicated that 15451-1 was a hypervirulent K. pneumoniae (hvKP) isolate. Conjugation experiments were conducted with rifampin-resistant E. coli EC600 (LacZ⁻ Nal^r Rif^r) as the recipient. Transconjugants were successfully obtained using both K. pneumoniae 15451-1 and E. coli 15451-2 (referred to here as 15451-1-C and 15451-2-C, respectively). Both the parental isolates and transconjugants were colistin resistant and carried mcr-1, as shown in Table 1. Plasmid analysis was carried out by S1-nuclease digestion and pulsed-field gel electrophoresis (PFGE) as previously described (12), and probing with the mcr-1 DNA fragment was subsequently performed. The mcr-1 gene was located on an ∼75-kb IncF plasmid in E. coli 15451-2; however, the identity of the mcr-1-carrying plasmid in K. pneumoniae remained unknown, as the band of this isolate always diffused during S1-PFGE analysis. Multilocus sequence type (MLST) analysis of K. pneumoniae and E. coli was conducted according to previous reports (13, 14). Strain 15451-1 and strain 15451-2 were identified as ST661 and ST3176, respectively; ST661 and ST3176 have seldom been reported in the literature, and neither belongs to the popular sequence types.

TABLE 1.

Characteristics of Klebsiella pneumoniae 15451-1, Escherichia coli 15451-2, corresponding transconjugants (15451-1-C and 15451-1-C), and the recipient E. coli isolate EC600

Isolate	Antimicrobial susceptibility (μg/ml)a																Resistance gene(s)
	AMP	AMC	CZO	FOX	CRO	FEP	ATM	ETP	IPM	AMK	GEN	TOB	CIP	TIG	F	CL
15451-1	≥32	≥32	≥64	≥64	≤1	≤1	≤1	≤0.5	≤1	≤2	≤1	8	2	2	128	16	mcr-1, blaDHA-1
15451-1-C	≥32	8	≤4	16	≤1	≤1	≤1	≤0.5	≤1	≤2	≤1	≤1	≤0.25	≤0.5	≤16	16	mcr-1, blaDHA-1
15451-2	16	8	≤4	32	≤1	≤1	≤1	≤0.5	≤1	≤2	≤1	≤1	0.5	≤0.5	≤16	8	mcr-1
15451-2-C	≥32	8	≤4	16	≤1	≤1	≤1	≤0.5	≤1	≤2	≤1	≤1	≤0.25	≤0.5	≤16	16	mcr-1
EC600	≤4	≤2	≤4	≤4	≤1	≤1	≤1	≤0.5	≤1	≤2	≤1	≤1	≤0.25	≤0.5	≤16	≤1

^aAMP, ampicillin; AMC, amoxicillin-clavulanic acid; CZO, cefazolin; FOX, cefoxitin; CRO, ceftriaxone; FEP, cefepime; ATM, aztreonam; ETP, ertapenem; IPM, imipenem; AMK, amikacin; GEN, gentamicin; TOB, tobramycin; CIP, ciprofloxacin; TIG, tigecycline; F, nitrofurantoin; CL, colistin.

Previous reports (5, 7) of high rates of detection of mcr-1 in animals and human strongly suggested an underlying route of food chain transmission of mcr-1. Interestingly, the patient in this study was about 1 year of age, fed on breast milk and rice flour, and had no history of contact with poultry or treatment with polymyxins. Therefore, the origin of the mcr-1-carrying isolates is unknown. Further inquiries revealed that, prior to the infant's hospitalization, the grandparents had suffered transient diarrhea, although because the family members declined to supply stool samples, further research was not possible.

Detection of mcr-1-carrying hvKP and E. coli isolates from an infant patient is of significant importance. First, hvKP isolates are increasingly reported, as they can cause life-threatening infections (15). The emergence of mcr-1 in hvKP isolates limits the available therapeutic options and presents an enormous challenge in the battle against infections. Second, the human gut is regarded as a reservoir for antibiotic resistance genes and plays a vital role in horizontal gene transfer. Simultaneous detection of mcr-1 in K. pneumoniae and E. coli isolates from one patient indicates horizontal transmission of mcr-1 between different species, leading to the likelihood of wide dissemination of colistin-resistant strains, and is thus a potential threat to the effective use of colistin. To address the global problem of polymyxin resistance, a comprehensive understanding of the emergence and epidemiology of these pathogens is needed. In addition, particular strategies, such as banning the use of polymyxins in agriculture and cutting off the transmission of resistant bacteria, should be promoted.