© 2014 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12260
APMIS 122: 1088–1095
Genotypic characteristics of multidrug-resistant Escherichia coli isolates associated with urinary tract infections XIAOLI CAO, ZHIFENG ZHANG, HAN SHEN, MINGZHE NING, JUNHAO CHEN, HONGXIA WEI and KUI ZHANG Department of Laboratory Medicine, The affiliated Nanjing Drum Tower Hospital of Nanjing University Medical School, Jiangsu, China
Cao X, Zhang Z, Shen H, Ning M, Chen J, Wei H, Zhang K. Genotypic characteristics of multidrug-resistant Escherichia coli isolates associated with urinary tract infections. APMIS 2014; 122: 1088–1095. Escherichia coli is an important pathogen involved in community-acquired urinary tract infections (CA-UTIs). In this study, we analyzed the prevalence of frequently occurring genes and the distribution of integrons in 51 multidrug-resistant (MDR) E. coli isolates associated with CA-UTIs. The clonality of these strains was investigated by phylogrouping, multi-locus sequence typing, and pulsed-field gel electrophoresis (PFGE). All these strains were found to produce two or more resistance determinants, ceftazidime-hydrolyzing CTX-M-type extended-spectrum b-lactamases (ESBLs) and plasmid-mediated quinolone resistance determinants were the most prevalent (92.2% and 51.0%, respectively). A sulfhydryl variable-61-producing E. coli strain was identified for the first time in China. The prevalence of class 1 integrons was 54.9%, class 2 integrons were detected in three isolates but no isolate contained a class 3 integron. Phylogenetic group D was the dominant, observed in 70.6% of the isolates. PFGE analysis revealed a high level of diversity. Twenty-four distinctive sequence types (STs) including four major STs (ST648, ST224, ST38, and ST405) were identified. To our knowledge, this is the first report on the characterization of MDR E. coli isolates associated with CA-UTIs in China; our results suggest that an MDR D-ST648 clone producing CTX-M-ESBLs has emerged as a major clone in the community setting. Key words: Multidrug resistance; PMQRs; ESCs; 16S-RMTase; CHbLs. Kui Zhang, Department of Laboratory Medicine, The affiliated Nanjing Drum Tower Hospital of Nanjing University Medical School, No. 321 Zhongshan Road, Nanjing 210008, China. e-mail: [email protected]
Multidrug-resistant (MDR) Escherichia coli strains are the leading causes of serious hospital-acquired and community-onset bacterial infections in health care settings (1), leading to high medical costs and high mortality rates (2). Generally, widely disseminated resistance determinants such as plasmid-mediated quinolone resistance determinants (PMQRs), exogenously acquired 16S rRNA methyltransferase (16SRMTase), and b-lactamases that include extendedspectrum b-lactamases (ESBLs), plasmid-mediated AmpC b-lactamases (pAmpCs), and carbapenemhydrolyzing b-lactamase (CHbLs) (3–6) are closely associated with the emergence of MDR E. coli, in addition to the over-expression of efflux pumps and decreased membrane permeability (2). Of these Received 16 September 2013. Accepted 7 January 2014
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resistance determinants, ESBLs such as CTX-M-14 and CTX-M-15 with greater activity against cefotaxime than other oxyimino-b-lactam substrates, are prevalent globally (7), followed by the variants of the sulfhydryl variable (SHV) and Temoniera (TEM) enzymes. AmpC b-lactamase genes are associated with resistance to cephamycins and oxyimino-b-lactams such as cefotaxime, with CMY-2 being the most prevalent pAmpC type (8). Plasmid-mediated quinolone resistance determinants such as the Qnr family genes (qnrA, qnrB, qnrC, qnrD, and qnrS), aac (6′)-Ib-cr, qepA, and oqxAB, are widely disseminated among Enterobacteriaceae (4, 9). The 16S-RMTase genes (i.e., armA, npmA, rmtA, rmtB, rmtC, rmtD, and rmtE) are widely distributed and have been reported from at least 30 countries or regions (5); they are responsible for the high level of resistance toward various
COMMUNITY-ACQUIRED MDR E. COLI ISOLATES
aminoglycosides. In addition, CHbLs have spread worldwide among Enterobacteriaceae spp. Most of them belong to the KPC, OXA-48, NDM, VIM, and IMP classes and have been reported in India, the United Kingdom, and European countries (3). Hospital-acquired infections caused by KPC- or OXA-48-producing Klebsiella pneumoniae isolates have been frequently reported (3). These resistance determinants are often located with plasmids, transposons, integrons, and insertion sequences, which mediate the transmission of resistance genes among Enterobacteriaceae (10, 11). Thus far, the prevalence of PMQRs among ESBLs-producing Enterobacteriaceae and the distribution of 16S-RMTase among PMQRs-producing strains have been investigated (12–15). However, there are little data available on the characterization of these determinants among the MDR E. coli isolates that are associated with communityacquired urinary tract infections (CA-UTIs). Hence, we aimed to investigate the prevalence of multiple resistance determinants and integrons among MDR E. coli isolates associated with CA-UTIs, as well as the clonality between these strains. MATERIALS AND METHODS Bacterial isolates A total of 323 consecutive nonrepetitive clinical E. coli urinary isolates were collected between April and July 2011 in Nanjing Drum Tower Hospital, Nanjing University, Jiangsu, China. Antimicrobial susceptibility testing of these strains by Kirby-Bauer’s disc diffusion method showed high frequencies of resistance toward fluoroquinolones (>70%), the oxyimino-cephalosporins (>40%) and aminoglycosides (>20%). Among the 268 isolates associated with CA-UTIs occurring in persons residing in the community, regardless of whether those persons have been receiving health care in an outpatient facility(16), 51 isolates simultaneously exhibiting resistance to cefotaxime (or ceftazidime), levofloxacin (or ciprofloxacin), and amikacin were used for further analysis. MacConkey agar plates containing 0.5 lg/mL meropenem were used for screening strains harboring CHbLs (17), and the minimum inhibitory concentrations of imipenem, meropenem, and ertapenem for the eight isolates with reduced susceptibility to meropenem were further determined by E-test strip (BioMerieux, Marcy L’Etoile, France).
Screening and identification of antimicrobial resistance genes DNA templates were prepared by the boiling method. All these isolates were screened for the presence of ESBLs including CTX, TEM, SHV, VEB, and OXA. Thirty-three isolates nonsusceptible to cefoxitin were analyzed for pAmpC (CMY-2-like, DHA, FOX, ACC-1, ACT/MIR, and CMY-1-like/MOX). Eight strains that survived on
© 2014 APMIS. Published by John Wiley & Sons Ltd
MacConkey agar plates containing 0.5 lg/mL meropenem were tested for CHbLs including KPC, OXA-48, IMP, VIM, NDM, DIM, SPM, and SIM (17). The presence of PMQRS genes (qnrA, qnrB, qnrC, qnrD, qnrS, qepA, aac (6′)Ib-cr, and oqxAB) as well as 16S rRNA methylation enzymes (ArmA, NpmA, RmtA, RmtB, RmtC, RmtD, and RmtE) were also analyzed for these strains. All of the antimicrobial resistance genes were amplified by polymerase chain reaction (PCR) as described previously (18–20). PCR products positive for resistance genes were further purified and sent to Majorbio Company for sequencing (Shanghai, China). Sequences were analyzed using the Chromas-Pro 1.7.5 application (Technelysium, Brisbane, Queensland, Australia) and BLAST Internet services (www. ncbi.nlm.nih.gov/BLAST).
Detection and characterization of integrons Integrons were detected by PCR amplification of the class I, II, III integrase specific int-1, int-2, and int-3 genes, as described previously (21). The variable region of integrons was further characterized by PCR and sequencing in all int-positive strains, and sequences were compared with those included in GenBank, to identify the gene cassettes.
Phylogrouping of the E. coli isolates These 51 E. coli strains were assigned to phylogenetic groups A, B1, B2, and D by triplex PCR developed by Clermont et al. (22). The PCR products were separated by 2.0% agarose gel electrophoresis. Strains were assigned into phylogenetic groups according to the protocol as follows (23): A (yjaA positive or yjaA, chuA, and TSPE4-C2 negative); B1 (TSPE4-C2 positive); B2 (chuA and yjaA positive or chuA, yjaA, and TSPE4-C2 positive); D (chuA positive or chuA and TSPE4-C2 positive).
Pulsed-field gel electrophoresis Pulsed-field gel electrophoresis (PFGE) was used to analyze the genomic relatedness among the 51 MDR E. coli isolates according to the CDC PulseNet protocol (24). Agaroseembedded bacterial genomic DNA was digested with the restriction enzyme XbaI (Fermentas, MBI, Darmstadt, Germany) at 37 °C for 2 h. Electrophoresis was performed on 1% agarose gel with 0.59 Tris-borate-EDTA buffer using a CHEF-DR III System (Bio-Rad Laboratories, Hercules, CA, USA). Running conditions consisted of 1 pulse time of 2.2–63.8 s for 19 h at 6 V/cm at a 120° angle. The PFGE profiles were compared using BioNumerics software version 6.6 (Applied Maths, Sint-Martens-Latem, Belgium), with the Pearson’s correlation coefficient for distance matrix and the UPGMA with optimization set at 1.5% to create the dendrogram. Cutoff line at 85% was used to analyze genetic relatedness.
Multilocus sequence typing All 51 MDR E. coli isolates in this study were further analyzed by multilocus sequence typing (MLST). Seven housekeeping genes, including adk, fumC, gyrB, icd, mdh, purA, and recA, were amplified and analyzed following the protocol available at http://mlst.ucc.ie/mlst/dbs/Ecoli.
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RESULTS Prevalence of resistance determinants
The prevalence of resistance determinants among the 51 MDR strains were displayed in Fig. 1. Determinants conferring resistance to b-lactam were identified in all isolates: CTX-M type b-lactamases were identified in 47/51 (92.2%), TEM-1b in 27/51(52.9%), OXA-1 in 16/51(31.4%), CMY-2 in 13/51 (25.5%), SHV-variants in 7/51(13.7%) and KPC-2 in 2/51 (3.9%). Reduced susceptibility to meropenem was present in 8 (15.7%) isolates (Table 1), two of them carried KPC-2 and the remaining six contained CMY-2 with five simultaneously producing CTX-M, indicating that AmpC (and CTX-M ESBL) enzymes in combination with decreased permeability may account for reduced susceptibility to meropenem (25, 26). Furthermore, a SHV-61-producing E. coli strain was identified for the first time in China. Among the 51 MDR E. coli isolates, 26 (51.0%) isolates carried at least 1 PMQR gene. Eleven (21.6%) isolates carried AAC(6′)-Ib-cr, 10 (19.7%) isolates carried Qnr variants including 5 QnrB4, 1 QnrB2, 3 QnrS1, and 1 QnrA1, 8 (15.7%) isolates carried OqxAB, and 3 (5.9%) isolates carried QepA. Genes conferring resistance to amikacin were detected as follows: 16S rRNA methylases (16SRMTase)-encoding genes were detected in 5/51 (9.8%) of isolates, aac(6′)1b-cr in 11/51 (21.6%) and aacA4 encoding aminoglycoside 6′-N-acetyltransferase (AAC(6′)-Ib) that confers resistance to amikacin and tobramycin in 24/51 (47.1%) of isolates, the latter being located within class 1 integrons. Additionally, aadA4, aadA5, and aadA2 conferring no resistance to amikacin were also identified. Collectively, all the 51 isolates carried two or more resistant determinants. The prevalence of PMQRs among ESBL-producing E. coli was found to be 24 (47.1%), 11 (21.6%) strains were co-producers of pAmpC and ESBLs, and 44 (86.3%) strains co-harbored b-lactamase genes. Furthermore, all TEM-1b- (52.9%), 16S-RMTase- (9.8%), and KPC- (3.9%)-producing isolates were CTX-Mpositive. In addition, the SHV-61 variant was identified in a CTX-M-14- and QnrB4-producing strain. Prevalence and characterization of integrons
The gene for class 1 integrase (intI1) was detected in 28 (54.9%) of 51 E. coli isolates tested, and the gene for class 2 integrase (intI2) was found in three isolates (Fig. 1). No intI3 was detected, and 2 (3.9%) isolates had both class 1 and class 2 1090
integrons. Molecular analysis revealed that class 1 integrons harbored four different antimicrobial resistance gene cassette arrays including aacA4catB8-aadA1 (17 strains), orfD-aacA4-catB8 (7 strains), dfrA17-aadA5 (3 strains), and dfrA12-orfFaadA2 (1 strains); and all the 3 class 2 integrons contained an identical cassette array comprising dfrA1-sat2-aadA1. These gene cassettes identified among class 1 and class 2 integrons in the collection have been frequently reported in E. coli isolates recovered from various sources (27). Phylogroups of the E. coli isolates
According to multi-PCR-based phylotyping, 36 (70.6%) strains belonged to phylogenetic group D. Additionally, 9 (17.6%) isolates belonged to group B2, 4 (7.8%) belonged to group A, and 2 (3.9%) belonged to group B1 (Fig. 1). Genetic relatedness
At the cutoff value of 85%, 41 unique PFGE types were observed for the 51 MDR E. coli isolates, as seen in the dendrogram (Fig. 1). And there was evident heterogeneity for the prevalent ST38 and ST405 clones. However, relatively homogenous PFGE profiles for the predominant sequence type (ST) clones of ST648 and ST224 were observed. Sequence types
Among the 51 isolates studied, MLST analysis identified 24 distinctive STs (Fig. 1). The predominant ST was ST648 (n = 9, 17.6%), followed by ST224 (n = 5, 9.8%), ST38 (n = 5, 9.8%), and ST405 (n = 4, 7.8%). These four majors STs accounted for 45.1% (n = 23) of all studied isolates. Among the other STs identified, seven STs including ST2437, ST3177, ST62, ST616, ST656, ST1410, and ST2003 have not previously been identified to be associated with MDR.
DISCUSSION Urinary tract infection is one of the most common bacterial infections, with E. coli being the cause of 80–90% of CA-UTIs (28). Thus, the increasing emergence and dissemination of MDR E. coli strains complicate the therapeutic management of infections, posing a serious challenge to public health. This study provided current data on the distribution of frequently prevalent resistance determinants and integrons as well as the genetic relatedness of 51 MDR E. coli isolates associated © 2014 APMIS. Published by John Wiley & Sons Ltd
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80
70
60
PFGE-Xba1
100
COMMUNITY-ACQUIRED MDR E. COLI ISOLATES
PFGE-Xba1
Key
PGs
STs
RTs
E799
D
2208
CTX-M-14,TEM-1b,SHV-1,RmtB,OqxAB
E812
D
2208
CTX-M-65,TEM-1b,SHV-11,CMY-2
E829
D
2208
CTX-M-14,QnrB4,QepA
E758
A
IntI1
2003
CTX-M-15,TEM-1b,OqxAB,QnrS1
E837
A
IntI1
2003
CTX-M-14,OXA-1
E775
A
2003
CTX-M-14,OXA-1
E848
B2
IntI1
1410
CTX-M-15,TEM-1b,KPC-2
E651
D
IntI1
1638
CTX-M-14,TEM-Ib,RmtB,QnrB2,OqxAB
E828
B1
IntI1
155
CTX-M-14,SHV-61,QnrB4
E777
D
393
DHA-1,QnrB4,QnrS1
E816
D
656
OXA-1,CMY-2
E624
B2
131
CTX-M-14,OXA-1
E852
B2
131
CTX-M-14,TEM-1b,QepA
E851
D
616
CTX-M-15,TEM-1b,KPC-2,AAC(6')-Ib-cr
E813
D
448
CTX-M-15,CMY-2,QnrB4,OqxAB
E644
D
IntI1
224
CTX-M-15,OXA-1,AAC(6')-Ib-cr
E742
D
IntI1
224
CTX-M-14,TEM-1b,CMY-2
E600
D
IntI1
224
CTX-M-14,TEM-1b
E708
D
IntI1
224
CTX-M-15,CMY-2,AAC(6')-Ib-cr
E638
D
IntI1,IntI2
224
CTX-M-15,AAC(6')-Ib-cr
E670
D
IntI1
405
CTX-M-14,TEM-1b,OXA-1
E831
D
405
SHV-12,OXA-1,AAC(6')-Ib-cr
E629
D
IntI1
405
CTX-M-14,OXA-1
E643
D
IntI1
405
CTX-M-15,TEM-1b,RmtB
E783
D
69
CTX-M-15,SHV-11
E858
D
62
CTX-M-14,TEM-1b,OXA-1
E518
A
IntI1
1284
CTX-M-14,TEM-1b,AAC(6')-Ib-cr
E529
B2
IntI1
1193
CTX-M-14,TEM-1b
E709
B1
IntI1,IntI2
101
CTX-M-15,OXA-1,QepA
E704
B2
73
CTX-M-27,OXA-1
E832
B2
73
CTX-M-14,OXA-1
E767
D
648
CTX-M-14,TEM-1b,OXA-1
E781
D
648
CTX-M-14,TEM-1b,OXA-1
E853
D
648
CTX-M-15,QnrB4,QnrS1
E563
D
648
CTX-M-14,AAC(6')-Ib-cr
E845
D
IntI1
648
CTX-M-14,TEM-1b,CMY-2,QnrA1,OqxAB
E790
D
IntI2
648
CTX-M-14,TEM-1b,CMY-2
E729
D
648
CTX-M-14,TEM-1b
E833
D
648
CMY-2,OqxAB,AAC(6')-Ib-cr
E662
D
IntI1
648
CTX-M-14,CMY-2,AAC(6')-Ib-cr
E743
D
IntI1
38
CTX-M-14,QepA
E840
D
38
CTX-M-14,OXA-1
E565
D
IntI1
38
CTX-M-14,TEM-1b
E612
D
IntI1
38
CTX-M-14,TEM-1b
E641
D
IntI1
38
CTX-M-14,TEM-1b
E645
B2
IntI1
410
CTX-M-15,TEM-Ib,CMY-2,AAC(6')-Ib-cr
E776
B2
IntI1
410
CTX-M-14,TEM-1b,RmtB,OqxAB
E728
D
IntI1
354
CTX-M-15,TEM-1b,ArmA,QnrB4
E782
D
IntI1
354
CTX-M-14,TEM-1b,SHV-11,CMY-2,OqxAB
E773
B2
2437
CTX-M-15,SHV-11,CMY-2,OqxAB,AAC(6')-Ib-cr
E788
D
3177
CTX-M-14,TEM-1b,OXA-1
Int
IntI1
IntI1
Fig. 1. Dendrogram based on pulse-field gel electrophoresis developed in BioNumerics for 51 multidrug-resistant Escherichia coli isolates associated with CA-UTIs. PGs, phylogroups; Int, integrons; STs, sequence types; RTs, resistance determinants.
with CA-UTIs. To our knowledge, this is the first study on the characterization of MDR E. coli isolates associated with CA-UTIs in mainland, China. © 2014 APMIS. Published by John Wiley & Sons Ltd
Our study showed a quite higher prevalence of CTX-M ESBLs among MDR E. coli strains than that in other countries such as Tunisia (29). In
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Table 1. Carbapenem MICs (lg/mL) determined by E-test for the 8 isolates with reduced susceptibility to meropenem Imipenem Meropenem Ertapenem E645 1 0.75 1.5 E742 2 2 4 E773 2 3 8 E812 4 0.75 6 E816 2 3 8 E845 4 2 8 E848 32 64 64 E851 32 32 64 MIC: minimum inhibitory concentrations.
addition, the prevalence of TEM-1b, OXA-1, 16SRMTase, SHV-variants, and KPC, accompanied by CTX-M ESBLs and the frequent occurrence of PMQRs among these CTX-M-producing E. coli isolates indicate that CTX-M-producing strains were more often MDR than bacteria producing other types of ESBLs. This may be due to the association between the blaCTX-M genes and a variety of mobile genetic elements mediating rapid and efficient cell-to-cell dissemination of high-risk multiresistant clones (30), which can be further demonstrated by the wide distribution of class I integrons in our MDR isolates. It is noteworthy that our study detected a SHV-61 variant in an E. coli isolate as SHV-61 was only identified in 1 K. pneumoniae strain after it was generated by a homology model in anin-silico study (31, 32). Although this study is consistent with the finding that ArmA and RmtB are the most prevalent 16S-RMTase among enterobacterial strains (5), the occurrence of these genes in our study was obviously lower than that among clinical MDR E. coli isolates collected from 2007 to 2009 (14). Thus, the high prevalence of AAC(6′)-Ib (encoded by aacA4 and associated with class I integrons) and AAC(6′)-Ib-cr in our study may play a crucial role in the amikacin resistance, in addition to the reduced intracellular concentration of aminoglycosides and mutation of 30S ribosomal subunit target (33). Additionally, as reported previously (8), CMY-2 is the most prevalent pAmpC mediating resistance to cephamycins in our study. However, the over-expression of chromosomal ampC resulting from multiple mutations in the ampC promoter region combining with altered expression of outer membrane proteins constituting porins may also contribute to the nonsusceptibilities of these strains to cefoxitin (34, 35). And the frequent occurrence of CMY-2 and CTX-M among the six strains with reduced susceptibility to meropenem indicates that high expression of AmpC or CTX-M ESBLs in combination with porin alterations plays a major role in increased carbapenem minimum inhibitory
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concentrations in the other six strains without producing KPC (25, 26, 31). To date, multiple resistance determinants cocarried by the same strain have been frequently identified in several enterobacterial isolates worldwide, especially in E. coli strains, namely, E. coli strains containing VIM, SHV-12, and QnrS from a Bulgarian Hospital (36); an E. coli isolate co-producing QepA1, CTX-M-15, and RmtB from Detroit, Michigan (37); and E. coli clinical isolates positive for CTX-M, AAC (6′)-Ib-cr, and QnrB from Rio de Janeiro, Brazil (38). However, the resistance determinants co-carried by some of our strains are quite different, such as the co-occurrence of CTX-M-15, SHV-11, CMY-2, OqxAB, and AAC (6′)-Ib-cr in strain E773 and the co-produced CTX-M-14, TEM-1b, QnrB2, RmtB, and OqxAB in strain E651. To the best of our knowledge, this is the first report of these co-carried determinants in E. coli strains. Of note, the identification of different resistance genes was very frequent within the same ST in our study. This may result from the mobilization of resistance genes by integrative and conjugative elements such as integrons or insertion sequence, which are highly mobile genetic vehicles for the transfer of resistance genes between species of bacteria (39). Our study found that phylogenetic group D was the dominant MDR E. coli isolates associated with CA-UTIs, indicating that group D strains may be prone to be more resistant to antimicrobials than group B2 strains. This is concordant with a previous report showing that PMQR-, 16S-RMTase-, and blactamase-producing isolates preferentially belonged to phylogroup D (40). However, phylogroup B2 E. coli isolates are the most frequent pathogens involved in UTIs and associated with multiple virulence and resistance genes in Europe and the United States (41, 42). The high diversity of the genetic relatedness between these 51 MDR isolates identified by PFGE suggested that mobile elements may play a major role in producing multidrug resistance by mediating the rapid spread of these resistance determinants under the selective pressure of improper or incomplete dosages of antibiotics (43). However, clone dissemination of dominant STs including ST648, ST38, and ST405 seems to play an additional role, and these clones have been frequently identified in clinical isolates derived from humans and are highly resistant to the majority of antimicrobial agents for clinical use (44, 45). Noteworthily, this is the first study to report the prevalence of MDR ST224 clones although ST224 producing KPC or NDM-1 has been previously reported in Europe and China (46, 47). In addition, the occurrence of only 2
© 2014 APMIS. Published by John Wiley & Sons Ltd
COMMUNITY-ACQUIRED MDR E. COLI ISOLATES
ST131 strains in our study is quite different from the widely disseminated B2-ST131 E. coli clone linked predominantly to the community-onset MDR-extra-intestinal infections all over the world (48). This may indicate that ST648, in addition to ST131, has been extensively disseminated throughout the community setting. Moreover, ST155, ST73, ST69, ST354, and ST656 have been reported, albeit relatively infrequently, in UTI-associated isolates (49, 50). To the best of our knowledge, other ST clones such as ST2437, ST3177, ST62, ST616, ST656, ST1410, and ST2003 have not been reported to be associated with MDR. Altogether, such diverse STs indicated that CA-UTIs might be caused by different E. coli clones. In conclusion, our study is the first one to characterize MDR E. coli isolates associated with CA-UTIs. We found that a great majority of MDR isolates belonged to ESBL-producing strains that also carried several other resistance determinants. Our data suggest that specific MDR ST clones of E. coli, such as ST648 and ST224, co-carrying multiple resistance determinants, have been spreading throughout the community setting. These E. coli isolates may be potential factors for regional dissemination of antibiotic resistance.
This study is supported by Nan Jing Medical Science and Technique Development Foundation (grant no. QRX11191).And we are very grateful to EU Reference Laboratory for antimicrobial resistance of National Food Institute of Technical University of Denmark for providing the positive controls for our experiments.
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© 2014 APMIS. Published by John Wiley & Sons Ltd
6. Swick MC, Morgan-Linnell SK, Carlson KM, Zechiedrich L. Expression of multidrug efflux pump genes acrAB-tolC, mdfA, and norE in Escherichia coli clinical isolates as a function of fluoroquinolone and multidrug resistance. Antimicrob Agents Chemother 2011;55:921–4. 7. Canton R, Gonzalez-Alba JM, Galan JC. CTX-M enzymes: origin and diffusion. Front Microbiol 2012;3:110. 8. Matsumura Y, Yamamoto M, Higuchi T, Komori T, Tsuboi F, Hayashi A, et al. Prevalence of plasmidmediated AmpC beta-lactamase-producing Escherichia coli and spread of the ST131 clone among extendedspectrum beta-lactamase-producing E. coli in Japan. Int J Antimicrob Agents 2012;40:158–62. 9. Schultsz C, Geerlings S. Plasmid-mediated resistance in Enterobacteriaceae: changing landscape and implications for therapy. Drugs 2012;72:1–16. 10. Stalder T, Barraud O, Casellas M, Dagot C, Ploy MC. Integron involvement in environmental spread of antibiotic resistance. Front Microbiol 2012;3:119. 11. Naiemi NA, Duim B, Savelkoul PH, Spanjaard L, de Jonge E, Bart A, et al. Widespread transfer of resistance genes between bacterial species in an intensive care unit: implications for hospital epidemiology. J Clin Microbiol 2005;43:4862–4. 12. Lin CJ, Siu LK, Ma L, Chang YT, Lu PL. Molecular epidemiology of ciprofloxacin-resistant extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in Taiwan. Microb Drug Resist 2012;18:52–8. 13. Yang HF, Cheng J, Hu LF, Ye Y, Li JB. Plasmidmediated quinolone resistance in extended-spectrumbeta-lactamase- and AmpC beta-lactamase-producing Serratia marcescens in China. Antimicrob Agents Chemother 2012;56:4529–31. 14. Yang J, Ye L, Wang W, Luo Y, Zhang Y, Han L. Diverse prevalence of 16S rRNA methylase genes armA and rmtB amongst clinical multidrug-resistant Escherichia coli and Klebsiella pneumoniae isolates. Int J Antimicrob Agents 2011;38:348–51. 15. Briales A, Rodriguez-Martinez JM, Velasco C, de Alba PD, Rodriguez-Bano J, Martinez-Martinez L, et al. Prevalence of plasmid-mediated quinolone resistance determinants qnr and aac(6′)-Ib-cr in Escherichia coli and Klebsiella pneumoniae producing extended-spectrum beta-lactamases in Spain. Int J Antimicrob Agents 2012;39:431–4. 16. Friedman ND, Kaye KS, Stout JE, McGarry SA, Trivette SL, Briggs JP, et al. Health care–associated bloodstream infections in adults: a reason to change the accepted definition of community-acquired infections. Ann Intern Med 2002;137:791–7. 17. Cohen Stuart J, Leverstein-Van Hall MA, Dutch Working Party on the Detection of Highly Resistant Microorganisms. Guideline for phenotypic screening and confirmation of carbapenemases in Enterobacteriaceae. Int J Antimicrob Agents 2010;36:205–10. 18. Dallenne C, Da Costa A, Decre D, Favier C, Arlet G. Development of a set of multiplex PCR assays for the detection of genes encoding important beta-lactamases in Enterobacteriaceae. J Antimicrob Chemother 2010;65:490–5. 19. Poirel L, Walsh TR, Cuvillier V, Nordmann P. Multiplex PCR for detection of acquired carbapenemase genes. Diagn Microbiol Infect Dis 2011;70:119–23.
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20. Doi Y, Arakawa Y. 16S ribosomal RNA methylation: emerging resistance mechanism against aminoglycosides. Clin Infect Dis 2007;45:88–94. 21. Mokracka J, Gruszczynska B, Kaznowski A. Integrons, beta-lactamase and qnr genes in multidrug resistant clinical isolates of Proteus mirabilis and P. vulgaris. APMIS 2012;120:950–8. 22. Clermont O, Bonacorsi S, Bingen E. Rapid and simple determination of the Escherichia coli phylogenetic group. Appl Environ Microbiol 2000;66:4555–8. 23. Gordon DM, Clermont O, Tolley H, Denamur E. Assigning Escherichia coli strains to phylogenetic groups: multi-locus sequence typing versus the PCR triplex method. Environ Microbiol 2008;10:2484–96. 24. Ribot EM, Fair MA, Gautom R, Cameron DN, Hunter SB, Swaminathan B, et al. Standardization of pulsed-field gel electrophoresis protocols for the subtyping of Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet. Foodborne Pathog Dis 2006;3:59–67. 25. Girlich D, Poirel L, Nordmann P. CTX-M expression and selection of ertapenem resistance in Klebsiella pneumoniae and Escherichia coli. Antimicrob Agents Chemother 2009;53:832–4. 26. Chia JH, Siu LK, Su LH, Lin HS, Kuo AJ, Lee MH, et al. Emergence of carbapenem-resistant Escherichia coli in Taiwan: resistance due to combined CMY-2 production and porin deficiency. J Chemother 2009;21:621–6. 27. Sun J, Zheng F, Wang F, Wu K, Wang Q, Chen Q, et al. Class 1 integrons in urinary isolates of extendedspectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae in Southern China during the past five years. Microb Drug Resist 2013;19:289–94. 28. Jones RN, Kugler KC, Pfaller MA, Winokur PL. Characteristics of pathogens causing urinary tract infections in hospitals in North America: results from the SENTRY Antimicrobial Surveillance Program, 1997. Diagn Microbiol Infect Dis 1999;35:55–63. 29. Hammami S, Saidani M, Ferjeni S, Aissa I, Slim A, Boutiba-Ben Boubaker I. Characterization of extended spectrum beta-lactamase-producing Escherichia coli in community-acquired urinary tract infections in Tunisia. Microb Drug Resist 2013;19:231–6. 30. D’Andrea MM, Arena F, Pallecchi L, Rossolini GM. CTX-M-type beta-lactamases: a successful story of antibiotic resistance. Int J Med Microbiol 2013;303:305–17. 31. Dahmen S, Mansour W, Charfi K, Boujaafar N, Arlet G, Bouallegue O. Imipenem resistance in Klebsiella pneumoniae is associated to the combination of plasmid-mediated CMY-4 AmpC beta-lactamase and loss of an outer membrane protein. Microb Drug Resist 2012;18:479–83. 32. Khan AU, Baig MH, Wadhwa G. Molecular docking analysis of new generation cephalosporins interactions with recently known SHV-variants. Bioinformation 2011;5:331–5. 33. Shakil S, Khan R, Zarrilli R, Khan AU. Aminoglycosides versus bacteria–a description of the action, resistance mechanism, and nosocomial battleground. J Biomed Sci 2008;15:5–14. 34. Tracz DM, Boyd DA, Hizon R, Bryce E, McGeer A, Ofner-Agostini M, et al. ampC gene expression in promoter mutants of cefoxitin-resistant Escherichia
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48. Totsika M, Beatson SA, Sarkar S, Phan MD, Petty NK, Bachmann N, et al. Insights into a multidrug resistant Escherichia coli pathogen of the globally disseminated ST131 lineage: genome analysis and virulence mechanisms. PLoS ONE 2011;6:e26578. 49. Galani I, Souli M, Panagea T, Poulakou G, Kanellakopoulou K, Giamarellou H. Prevalence of 16S rRNA methylase genes in Enterobacteriaceae isolates from a
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Greek university hospital. Clin Microbiol Infect 2012;18:E52–4. 50. Harada K, Nakai Y, Kataoka Y. Mechanisms of resistance to cephalosporin and emergence of O25bST131 clone harboring CTX-M-27 beta-lactamase in extraintestinal pathogenic Escherichia coli from dogs and cats in Japan. Microbiol Immunol 2012;56: 480–5.
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Genotypic characteristics of multidrug-resistant Escherichia coli isolates associated with urinary tract infections XIAOLI CAO, ZHIFENG ZHANG, HAN SHEN, MINGZHE NING, JUNHAO CHEN, HONGXIA WEI and KUI ZHANG Department of Laboratory Medicine, The affiliated Nanjing Drum Tower Hospital of Nanjing University Medical School, Jiangsu, China
Cao X, Zhang Z, Shen H, Ning M, Chen J, Wei H, Zhang K. Genotypic characteristics of multidrug-resistant Escherichia coli isolates associated with urinary tract infections. APMIS 2014; 122: 1088–1095. Escherichia coli is an important pathogen involved in community-acquired urinary tract infections (CA-UTIs). In this study, we analyzed the prevalence of frequently occurring genes and the distribution of integrons in 51 multidrug-resistant (MDR) E. coli isolates associated with CA-UTIs. The clonality of these strains was investigated by phylogrouping, multi-locus sequence typing, and pulsed-field gel electrophoresis (PFGE). All these strains were found to produce two or more resistance determinants, ceftazidime-hydrolyzing CTX-M-type extended-spectrum b-lactamases (ESBLs) and plasmid-mediated quinolone resistance determinants were the most prevalent (92.2% and 51.0%, respectively). A sulfhydryl variable-61-producing E. coli strain was identified for the first time in China. The prevalence of class 1 integrons was 54.9%, class 2 integrons were detected in three isolates but no isolate contained a class 3 integron. Phylogenetic group D was the dominant, observed in 70.6% of the isolates. PFGE analysis revealed a high level of diversity. Twenty-four distinctive sequence types (STs) including four major STs (ST648, ST224, ST38, and ST405) were identified. To our knowledge, this is the first report on the characterization of MDR E. coli isolates associated with CA-UTIs in China; our results suggest that an MDR D-ST648 clone producing CTX-M-ESBLs has emerged as a major clone in the community setting. Key words: Multidrug resistance; PMQRs; ESCs; 16S-RMTase; CHbLs. Kui Zhang, Department of Laboratory Medicine, The affiliated Nanjing Drum Tower Hospital of Nanjing University Medical School, No. 321 Zhongshan Road, Nanjing 210008, China. e-mail: [email protected]
Multidrug-resistant (MDR) Escherichia coli strains are the leading causes of serious hospital-acquired and community-onset bacterial infections in health care settings (1), leading to high medical costs and high mortality rates (2). Generally, widely disseminated resistance determinants such as plasmid-mediated quinolone resistance determinants (PMQRs), exogenously acquired 16S rRNA methyltransferase (16SRMTase), and b-lactamases that include extendedspectrum b-lactamases (ESBLs), plasmid-mediated AmpC b-lactamases (pAmpCs), and carbapenemhydrolyzing b-lactamase (CHbLs) (3–6) are closely associated with the emergence of MDR E. coli, in addition to the over-expression of efflux pumps and decreased membrane permeability (2). Of these Received 16 September 2013. Accepted 7 January 2014
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resistance determinants, ESBLs such as CTX-M-14 and CTX-M-15 with greater activity against cefotaxime than other oxyimino-b-lactam substrates, are prevalent globally (7), followed by the variants of the sulfhydryl variable (SHV) and Temoniera (TEM) enzymes. AmpC b-lactamase genes are associated with resistance to cephamycins and oxyimino-b-lactams such as cefotaxime, with CMY-2 being the most prevalent pAmpC type (8). Plasmid-mediated quinolone resistance determinants such as the Qnr family genes (qnrA, qnrB, qnrC, qnrD, and qnrS), aac (6′)-Ib-cr, qepA, and oqxAB, are widely disseminated among Enterobacteriaceae (4, 9). The 16S-RMTase genes (i.e., armA, npmA, rmtA, rmtB, rmtC, rmtD, and rmtE) are widely distributed and have been reported from at least 30 countries or regions (5); they are responsible for the high level of resistance toward various
COMMUNITY-ACQUIRED MDR E. COLI ISOLATES
aminoglycosides. In addition, CHbLs have spread worldwide among Enterobacteriaceae spp. Most of them belong to the KPC, OXA-48, NDM, VIM, and IMP classes and have been reported in India, the United Kingdom, and European countries (3). Hospital-acquired infections caused by KPC- or OXA-48-producing Klebsiella pneumoniae isolates have been frequently reported (3). These resistance determinants are often located with plasmids, transposons, integrons, and insertion sequences, which mediate the transmission of resistance genes among Enterobacteriaceae (10, 11). Thus far, the prevalence of PMQRs among ESBLs-producing Enterobacteriaceae and the distribution of 16S-RMTase among PMQRs-producing strains have been investigated (12–15). However, there are little data available on the characterization of these determinants among the MDR E. coli isolates that are associated with communityacquired urinary tract infections (CA-UTIs). Hence, we aimed to investigate the prevalence of multiple resistance determinants and integrons among MDR E. coli isolates associated with CA-UTIs, as well as the clonality between these strains. MATERIALS AND METHODS Bacterial isolates A total of 323 consecutive nonrepetitive clinical E. coli urinary isolates were collected between April and July 2011 in Nanjing Drum Tower Hospital, Nanjing University, Jiangsu, China. Antimicrobial susceptibility testing of these strains by Kirby-Bauer’s disc diffusion method showed high frequencies of resistance toward fluoroquinolones (>70%), the oxyimino-cephalosporins (>40%) and aminoglycosides (>20%). Among the 268 isolates associated with CA-UTIs occurring in persons residing in the community, regardless of whether those persons have been receiving health care in an outpatient facility(16), 51 isolates simultaneously exhibiting resistance to cefotaxime (or ceftazidime), levofloxacin (or ciprofloxacin), and amikacin were used for further analysis. MacConkey agar plates containing 0.5 lg/mL meropenem were used for screening strains harboring CHbLs (17), and the minimum inhibitory concentrations of imipenem, meropenem, and ertapenem for the eight isolates with reduced susceptibility to meropenem were further determined by E-test strip (BioMerieux, Marcy L’Etoile, France).
Screening and identification of antimicrobial resistance genes DNA templates were prepared by the boiling method. All these isolates were screened for the presence of ESBLs including CTX, TEM, SHV, VEB, and OXA. Thirty-three isolates nonsusceptible to cefoxitin were analyzed for pAmpC (CMY-2-like, DHA, FOX, ACC-1, ACT/MIR, and CMY-1-like/MOX). Eight strains that survived on
© 2014 APMIS. Published by John Wiley & Sons Ltd
MacConkey agar plates containing 0.5 lg/mL meropenem were tested for CHbLs including KPC, OXA-48, IMP, VIM, NDM, DIM, SPM, and SIM (17). The presence of PMQRS genes (qnrA, qnrB, qnrC, qnrD, qnrS, qepA, aac (6′)Ib-cr, and oqxAB) as well as 16S rRNA methylation enzymes (ArmA, NpmA, RmtA, RmtB, RmtC, RmtD, and RmtE) were also analyzed for these strains. All of the antimicrobial resistance genes were amplified by polymerase chain reaction (PCR) as described previously (18–20). PCR products positive for resistance genes were further purified and sent to Majorbio Company for sequencing (Shanghai, China). Sequences were analyzed using the Chromas-Pro 1.7.5 application (Technelysium, Brisbane, Queensland, Australia) and BLAST Internet services (www. ncbi.nlm.nih.gov/BLAST).
Detection and characterization of integrons Integrons were detected by PCR amplification of the class I, II, III integrase specific int-1, int-2, and int-3 genes, as described previously (21). The variable region of integrons was further characterized by PCR and sequencing in all int-positive strains, and sequences were compared with those included in GenBank, to identify the gene cassettes.
Phylogrouping of the E. coli isolates These 51 E. coli strains were assigned to phylogenetic groups A, B1, B2, and D by triplex PCR developed by Clermont et al. (22). The PCR products were separated by 2.0% agarose gel electrophoresis. Strains were assigned into phylogenetic groups according to the protocol as follows (23): A (yjaA positive or yjaA, chuA, and TSPE4-C2 negative); B1 (TSPE4-C2 positive); B2 (chuA and yjaA positive or chuA, yjaA, and TSPE4-C2 positive); D (chuA positive or chuA and TSPE4-C2 positive).
Pulsed-field gel electrophoresis Pulsed-field gel electrophoresis (PFGE) was used to analyze the genomic relatedness among the 51 MDR E. coli isolates according to the CDC PulseNet protocol (24). Agaroseembedded bacterial genomic DNA was digested with the restriction enzyme XbaI (Fermentas, MBI, Darmstadt, Germany) at 37 °C for 2 h. Electrophoresis was performed on 1% agarose gel with 0.59 Tris-borate-EDTA buffer using a CHEF-DR III System (Bio-Rad Laboratories, Hercules, CA, USA). Running conditions consisted of 1 pulse time of 2.2–63.8 s for 19 h at 6 V/cm at a 120° angle. The PFGE profiles were compared using BioNumerics software version 6.6 (Applied Maths, Sint-Martens-Latem, Belgium), with the Pearson’s correlation coefficient for distance matrix and the UPGMA with optimization set at 1.5% to create the dendrogram. Cutoff line at 85% was used to analyze genetic relatedness.
Multilocus sequence typing All 51 MDR E. coli isolates in this study were further analyzed by multilocus sequence typing (MLST). Seven housekeeping genes, including adk, fumC, gyrB, icd, mdh, purA, and recA, were amplified and analyzed following the protocol available at http://mlst.ucc.ie/mlst/dbs/Ecoli.
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RESULTS Prevalence of resistance determinants
The prevalence of resistance determinants among the 51 MDR strains were displayed in Fig. 1. Determinants conferring resistance to b-lactam were identified in all isolates: CTX-M type b-lactamases were identified in 47/51 (92.2%), TEM-1b in 27/51(52.9%), OXA-1 in 16/51(31.4%), CMY-2 in 13/51 (25.5%), SHV-variants in 7/51(13.7%) and KPC-2 in 2/51 (3.9%). Reduced susceptibility to meropenem was present in 8 (15.7%) isolates (Table 1), two of them carried KPC-2 and the remaining six contained CMY-2 with five simultaneously producing CTX-M, indicating that AmpC (and CTX-M ESBL) enzymes in combination with decreased permeability may account for reduced susceptibility to meropenem (25, 26). Furthermore, a SHV-61-producing E. coli strain was identified for the first time in China. Among the 51 MDR E. coli isolates, 26 (51.0%) isolates carried at least 1 PMQR gene. Eleven (21.6%) isolates carried AAC(6′)-Ib-cr, 10 (19.7%) isolates carried Qnr variants including 5 QnrB4, 1 QnrB2, 3 QnrS1, and 1 QnrA1, 8 (15.7%) isolates carried OqxAB, and 3 (5.9%) isolates carried QepA. Genes conferring resistance to amikacin were detected as follows: 16S rRNA methylases (16SRMTase)-encoding genes were detected in 5/51 (9.8%) of isolates, aac(6′)1b-cr in 11/51 (21.6%) and aacA4 encoding aminoglycoside 6′-N-acetyltransferase (AAC(6′)-Ib) that confers resistance to amikacin and tobramycin in 24/51 (47.1%) of isolates, the latter being located within class 1 integrons. Additionally, aadA4, aadA5, and aadA2 conferring no resistance to amikacin were also identified. Collectively, all the 51 isolates carried two or more resistant determinants. The prevalence of PMQRs among ESBL-producing E. coli was found to be 24 (47.1%), 11 (21.6%) strains were co-producers of pAmpC and ESBLs, and 44 (86.3%) strains co-harbored b-lactamase genes. Furthermore, all TEM-1b- (52.9%), 16S-RMTase- (9.8%), and KPC- (3.9%)-producing isolates were CTX-Mpositive. In addition, the SHV-61 variant was identified in a CTX-M-14- and QnrB4-producing strain. Prevalence and characterization of integrons
The gene for class 1 integrase (intI1) was detected in 28 (54.9%) of 51 E. coli isolates tested, and the gene for class 2 integrase (intI2) was found in three isolates (Fig. 1). No intI3 was detected, and 2 (3.9%) isolates had both class 1 and class 2 1090
integrons. Molecular analysis revealed that class 1 integrons harbored four different antimicrobial resistance gene cassette arrays including aacA4catB8-aadA1 (17 strains), orfD-aacA4-catB8 (7 strains), dfrA17-aadA5 (3 strains), and dfrA12-orfFaadA2 (1 strains); and all the 3 class 2 integrons contained an identical cassette array comprising dfrA1-sat2-aadA1. These gene cassettes identified among class 1 and class 2 integrons in the collection have been frequently reported in E. coli isolates recovered from various sources (27). Phylogroups of the E. coli isolates
According to multi-PCR-based phylotyping, 36 (70.6%) strains belonged to phylogenetic group D. Additionally, 9 (17.6%) isolates belonged to group B2, 4 (7.8%) belonged to group A, and 2 (3.9%) belonged to group B1 (Fig. 1). Genetic relatedness
At the cutoff value of 85%, 41 unique PFGE types were observed for the 51 MDR E. coli isolates, as seen in the dendrogram (Fig. 1). And there was evident heterogeneity for the prevalent ST38 and ST405 clones. However, relatively homogenous PFGE profiles for the predominant sequence type (ST) clones of ST648 and ST224 were observed. Sequence types
Among the 51 isolates studied, MLST analysis identified 24 distinctive STs (Fig. 1). The predominant ST was ST648 (n = 9, 17.6%), followed by ST224 (n = 5, 9.8%), ST38 (n = 5, 9.8%), and ST405 (n = 4, 7.8%). These four majors STs accounted for 45.1% (n = 23) of all studied isolates. Among the other STs identified, seven STs including ST2437, ST3177, ST62, ST616, ST656, ST1410, and ST2003 have not previously been identified to be associated with MDR.
DISCUSSION Urinary tract infection is one of the most common bacterial infections, with E. coli being the cause of 80–90% of CA-UTIs (28). Thus, the increasing emergence and dissemination of MDR E. coli strains complicate the therapeutic management of infections, posing a serious challenge to public health. This study provided current data on the distribution of frequently prevalent resistance determinants and integrons as well as the genetic relatedness of 51 MDR E. coli isolates associated © 2014 APMIS. Published by John Wiley & Sons Ltd
90
80
70
60
PFGE-Xba1
100
COMMUNITY-ACQUIRED MDR E. COLI ISOLATES
PFGE-Xba1
Key
PGs
STs
RTs
E799
D
2208
CTX-M-14,TEM-1b,SHV-1,RmtB,OqxAB
E812
D
2208
CTX-M-65,TEM-1b,SHV-11,CMY-2
E829
D
2208
CTX-M-14,QnrB4,QepA
E758
A
IntI1
2003
CTX-M-15,TEM-1b,OqxAB,QnrS1
E837
A
IntI1
2003
CTX-M-14,OXA-1
E775
A
2003
CTX-M-14,OXA-1
E848
B2
IntI1
1410
CTX-M-15,TEM-1b,KPC-2
E651
D
IntI1
1638
CTX-M-14,TEM-Ib,RmtB,QnrB2,OqxAB
E828
B1
IntI1
155
CTX-M-14,SHV-61,QnrB4
E777
D
393
DHA-1,QnrB4,QnrS1
E816
D
656
OXA-1,CMY-2
E624
B2
131
CTX-M-14,OXA-1
E852
B2
131
CTX-M-14,TEM-1b,QepA
E851
D
616
CTX-M-15,TEM-1b,KPC-2,AAC(6')-Ib-cr
E813
D
448
CTX-M-15,CMY-2,QnrB4,OqxAB
E644
D
IntI1
224
CTX-M-15,OXA-1,AAC(6')-Ib-cr
E742
D
IntI1
224
CTX-M-14,TEM-1b,CMY-2
E600
D
IntI1
224
CTX-M-14,TEM-1b
E708
D
IntI1
224
CTX-M-15,CMY-2,AAC(6')-Ib-cr
E638
D
IntI1,IntI2
224
CTX-M-15,AAC(6')-Ib-cr
E670
D
IntI1
405
CTX-M-14,TEM-1b,OXA-1
E831
D
405
SHV-12,OXA-1,AAC(6')-Ib-cr
E629
D
IntI1
405
CTX-M-14,OXA-1
E643
D
IntI1
405
CTX-M-15,TEM-1b,RmtB
E783
D
69
CTX-M-15,SHV-11
E858
D
62
CTX-M-14,TEM-1b,OXA-1
E518
A
IntI1
1284
CTX-M-14,TEM-1b,AAC(6')-Ib-cr
E529
B2
IntI1
1193
CTX-M-14,TEM-1b
E709
B1
IntI1,IntI2
101
CTX-M-15,OXA-1,QepA
E704
B2
73
CTX-M-27,OXA-1
E832
B2
73
CTX-M-14,OXA-1
E767
D
648
CTX-M-14,TEM-1b,OXA-1
E781
D
648
CTX-M-14,TEM-1b,OXA-1
E853
D
648
CTX-M-15,QnrB4,QnrS1
E563
D
648
CTX-M-14,AAC(6')-Ib-cr
E845
D
IntI1
648
CTX-M-14,TEM-1b,CMY-2,QnrA1,OqxAB
E790
D
IntI2
648
CTX-M-14,TEM-1b,CMY-2
E729
D
648
CTX-M-14,TEM-1b
E833
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CMY-2,OqxAB,AAC(6')-Ib-cr
E662
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CTX-M-14,CMY-2,AAC(6')-Ib-cr
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D
IntI1
38
CTX-M-14,QepA
E840
D
38
CTX-M-14,OXA-1
E565
D
IntI1
38
CTX-M-14,TEM-1b
E612
D
IntI1
38
CTX-M-14,TEM-1b
E641
D
IntI1
38
CTX-M-14,TEM-1b
E645
B2
IntI1
410
CTX-M-15,TEM-Ib,CMY-2,AAC(6')-Ib-cr
E776
B2
IntI1
410
CTX-M-14,TEM-1b,RmtB,OqxAB
E728
D
IntI1
354
CTX-M-15,TEM-1b,ArmA,QnrB4
E782
D
IntI1
354
CTX-M-14,TEM-1b,SHV-11,CMY-2,OqxAB
E773
B2
2437
CTX-M-15,SHV-11,CMY-2,OqxAB,AAC(6')-Ib-cr
E788
D
3177
CTX-M-14,TEM-1b,OXA-1
Int
IntI1
IntI1
Fig. 1. Dendrogram based on pulse-field gel electrophoresis developed in BioNumerics for 51 multidrug-resistant Escherichia coli isolates associated with CA-UTIs. PGs, phylogroups; Int, integrons; STs, sequence types; RTs, resistance determinants.
with CA-UTIs. To our knowledge, this is the first study on the characterization of MDR E. coli isolates associated with CA-UTIs in mainland, China. © 2014 APMIS. Published by John Wiley & Sons Ltd
Our study showed a quite higher prevalence of CTX-M ESBLs among MDR E. coli strains than that in other countries such as Tunisia (29). In
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Table 1. Carbapenem MICs (lg/mL) determined by E-test for the 8 isolates with reduced susceptibility to meropenem Imipenem Meropenem Ertapenem E645 1 0.75 1.5 E742 2 2 4 E773 2 3 8 E812 4 0.75 6 E816 2 3 8 E845 4 2 8 E848 32 64 64 E851 32 32 64 MIC: minimum inhibitory concentrations.
addition, the prevalence of TEM-1b, OXA-1, 16SRMTase, SHV-variants, and KPC, accompanied by CTX-M ESBLs and the frequent occurrence of PMQRs among these CTX-M-producing E. coli isolates indicate that CTX-M-producing strains were more often MDR than bacteria producing other types of ESBLs. This may be due to the association between the blaCTX-M genes and a variety of mobile genetic elements mediating rapid and efficient cell-to-cell dissemination of high-risk multiresistant clones (30), which can be further demonstrated by the wide distribution of class I integrons in our MDR isolates. It is noteworthy that our study detected a SHV-61 variant in an E. coli isolate as SHV-61 was only identified in 1 K. pneumoniae strain after it was generated by a homology model in anin-silico study (31, 32). Although this study is consistent with the finding that ArmA and RmtB are the most prevalent 16S-RMTase among enterobacterial strains (5), the occurrence of these genes in our study was obviously lower than that among clinical MDR E. coli isolates collected from 2007 to 2009 (14). Thus, the high prevalence of AAC(6′)-Ib (encoded by aacA4 and associated with class I integrons) and AAC(6′)-Ib-cr in our study may play a crucial role in the amikacin resistance, in addition to the reduced intracellular concentration of aminoglycosides and mutation of 30S ribosomal subunit target (33). Additionally, as reported previously (8), CMY-2 is the most prevalent pAmpC mediating resistance to cephamycins in our study. However, the over-expression of chromosomal ampC resulting from multiple mutations in the ampC promoter region combining with altered expression of outer membrane proteins constituting porins may also contribute to the nonsusceptibilities of these strains to cefoxitin (34, 35). And the frequent occurrence of CMY-2 and CTX-M among the six strains with reduced susceptibility to meropenem indicates that high expression of AmpC or CTX-M ESBLs in combination with porin alterations plays a major role in increased carbapenem minimum inhibitory
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concentrations in the other six strains without producing KPC (25, 26, 31). To date, multiple resistance determinants cocarried by the same strain have been frequently identified in several enterobacterial isolates worldwide, especially in E. coli strains, namely, E. coli strains containing VIM, SHV-12, and QnrS from a Bulgarian Hospital (36); an E. coli isolate co-producing QepA1, CTX-M-15, and RmtB from Detroit, Michigan (37); and E. coli clinical isolates positive for CTX-M, AAC (6′)-Ib-cr, and QnrB from Rio de Janeiro, Brazil (38). However, the resistance determinants co-carried by some of our strains are quite different, such as the co-occurrence of CTX-M-15, SHV-11, CMY-2, OqxAB, and AAC (6′)-Ib-cr in strain E773 and the co-produced CTX-M-14, TEM-1b, QnrB2, RmtB, and OqxAB in strain E651. To the best of our knowledge, this is the first report of these co-carried determinants in E. coli strains. Of note, the identification of different resistance genes was very frequent within the same ST in our study. This may result from the mobilization of resistance genes by integrative and conjugative elements such as integrons or insertion sequence, which are highly mobile genetic vehicles for the transfer of resistance genes between species of bacteria (39). Our study found that phylogenetic group D was the dominant MDR E. coli isolates associated with CA-UTIs, indicating that group D strains may be prone to be more resistant to antimicrobials than group B2 strains. This is concordant with a previous report showing that PMQR-, 16S-RMTase-, and blactamase-producing isolates preferentially belonged to phylogroup D (40). However, phylogroup B2 E. coli isolates are the most frequent pathogens involved in UTIs and associated with multiple virulence and resistance genes in Europe and the United States (41, 42). The high diversity of the genetic relatedness between these 51 MDR isolates identified by PFGE suggested that mobile elements may play a major role in producing multidrug resistance by mediating the rapid spread of these resistance determinants under the selective pressure of improper or incomplete dosages of antibiotics (43). However, clone dissemination of dominant STs including ST648, ST38, and ST405 seems to play an additional role, and these clones have been frequently identified in clinical isolates derived from humans and are highly resistant to the majority of antimicrobial agents for clinical use (44, 45). Noteworthily, this is the first study to report the prevalence of MDR ST224 clones although ST224 producing KPC or NDM-1 has been previously reported in Europe and China (46, 47). In addition, the occurrence of only 2
© 2014 APMIS. Published by John Wiley & Sons Ltd
COMMUNITY-ACQUIRED MDR E. COLI ISOLATES
ST131 strains in our study is quite different from the widely disseminated B2-ST131 E. coli clone linked predominantly to the community-onset MDR-extra-intestinal infections all over the world (48). This may indicate that ST648, in addition to ST131, has been extensively disseminated throughout the community setting. Moreover, ST155, ST73, ST69, ST354, and ST656 have been reported, albeit relatively infrequently, in UTI-associated isolates (49, 50). To the best of our knowledge, other ST clones such as ST2437, ST3177, ST62, ST616, ST656, ST1410, and ST2003 have not been reported to be associated with MDR. Altogether, such diverse STs indicated that CA-UTIs might be caused by different E. coli clones. In conclusion, our study is the first one to characterize MDR E. coli isolates associated with CA-UTIs. We found that a great majority of MDR isolates belonged to ESBL-producing strains that also carried several other resistance determinants. Our data suggest that specific MDR ST clones of E. coli, such as ST648 and ST224, co-carrying multiple resistance determinants, have been spreading throughout the community setting. These E. coli isolates may be potential factors for regional dissemination of antibiotic resistance.
This study is supported by Nan Jing Medical Science and Technique Development Foundation (grant no. QRX11191).And we are very grateful to EU Reference Laboratory for antimicrobial resistance of National Food Institute of Technical University of Denmark for providing the positive controls for our experiments.
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