Prevalence and drug resistance characteristics of carbapenem-resistant Enterobacteriaceae in Hangzhou, China

Abstract

With the abuse of antimicrobial agents in developing countries, increasing number of carbapenem-resistant Enterobacteriaceae (CRE) attracted considerable public concern. A retrospective study was conducted based on 242 CRE strains from a tertiary hospital in Hangzhou, China to investigate prevalence and drug resistance characteristics of CRE in southeast China. Bacterial species were identified. Antimicrobial susceptibility was examined by broth microdilution method or epsilometer test. Resistant β-lactamase genes were identified by polymerase chain reaction and sequencing. Genotypes were investigated by phylogenetic analysis. Klebsiella pneumoniae and Escherichia coli were the most prevalent types of species, with occurrence in 71.9% and 21.9% of the strains, respectively. All strains exhibited high resistance (>70%) against β-lactam antibiotics, ciprofloxacin, trimethoprim–sulfamethoxazole, and nitrofurantoin but exhibited low resistance against tigecycline (0.8%) and minocycline (8.3%). A total of 123 strains harbored more than two kinds of β-lactamase genes. bla_KPC-2, bla_SHV-11, bla_TEM-1, and bla_CTX-M-14 were the predominant genotypes, with detection rates of 60.3%, 61.6%, 43.4%, and 16.5%, respectively, and were highly identical with reference sequences in different countries, indicating potential horizontal dissemination. IMP-4 was the most frequent class B metallo-lactamases in this study. In conclusion, continuous surveillance and effective prevention should be emphasized to reduce spread of CRE.

Keywords

Enterobacteriaceae / carbapenem / β-lactamase genes / phylogenetic analysis

Introduction

Carbapenems represent a critical reserve class of β-lactam antimicrobials for multidrug-resistant Gram-negative bacterial infections and are often considered “last-line agents”; they possess the broadest spectrum of activity and greatest antibacterial potency [¹]. However, infections caused by carbapenem-resistant Enterobacteriaceae (CRE) are associated with high morbidity and mortality as few therapeutic options are available. In the past decade, infections caused by CRE were increasingly reported worldwide, such as in Asia, USA, Israel, and Lebanon [²–⁶]. A previously published meta-analysis study on epidemiology of CRE showed a stably escalating trend in Asia during 2000–2012 [⁶]. In China, prevalence of CRE showed a consistent trend. Surveillance of antibiotic resistance from China-CHINET showed that imipenem and meropenem resistance of Klebsiella spp. in 2005 reached 2.9% and 2.8%, respectively, whereas that in 2011 increased to 9.3% and 9.4%, respectively [⁷]. As described previously, the major resistance mechanism of CRE is associated with acquisition of carbapenemase genes or expression of high-level extended spectrum β-lactamases (ESBLs) or AmpC enzymes combined with mutation of porin, which decreases permeability to carbapenems [⁸].

This study aimed to detect carbapenemases, ESBLs, and AmpC in 242 CRE strains isolated from clinical specimens in a tertiary hospital in Hangzhou, China from January 2010 to June 2014, to investigate the prevalence of CRE and related resistant β-lactamase genes, and to provide basis for prevention and control of nosocomial infections.

Materials and methods

Bacterial strains

Between January 2010 and June 2014, 242 clinical CRE strains were isolated from clinical specimens that were collected from a tertiary hospital in Hangzhou, China. Duplicate isolates were excluded. CRE strain was defined as resistance to ertapenem or imipenem. Bacterial strains were identified at species level by standard microbiological methods using the Siemens MicroScan WalkAway 40 Plus Microbiology System (Siemens, Newark, DE, USA). Well-characterized strains were used as quality control strains in this study and listed as follows: Escherichia coli ATCC 25922 obtained from China CDC as a susceptible strain, Klebsiella pneumoniae KPC-2 strain for bla_KPC-2 (provided by Prof. Rong Zhang, Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China), Enterobacter cloacae IMI-1 strain for bla_IMI-1 (provided by Prof. A. A. Medeiros, The Miriam Hospital, Providence, RI, USA) [⁹], and K. pneumoniae IMP-4 strain for bla_IMP-4 (provided by Prof. G. A. Jacoby, Massachusetts General Hospital, Boston, MA, USA).

Antimicrobial susceptibility testing

Antimicrobial susceptibility was determined by broth microdilution method using the Microbiology System mentioned previously and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) 2015 guidelines [¹⁰] for all antimicrobial agents, except tigecycline, which was determined according to the breakpoint approved by the US Food and Drug Administration, as provided in the package insert (Tygacil® Product Insert; http://www.pfizerpro.com/hcp/tygacil). Tested antimicrobial agents included amikacin, tobramycin, tetracycline, trimethoprim–sulfamethoxazole, ciprofloxacin, cefoxitin, cefepime, ceftazidime, imipenem, aztreonam, ertapenem, amoxicillin clavulanate, and cefotaxime. Tigecycline, minocycline, and nitrofurantoin susceptibilities were evaluated by epsilometer test following manufacturer’s instructions (BioMerieux, Shanghai, China).

Carba NP confirmatory test (CNP test)

Results of CNP test were evaluated according to the CLSI 2015 guidelines [¹⁰].

Molecular analysis of resistance-related genes among CRE strains

All CRE strains were subjected to genetic analysis to investigate the carbapenem-resistant mechanism. Crude genomic DNA of strains for polymerase chain reaction (PCR) was extracted by heat lysis method [¹¹].

During PCR, previously published specific pairs of primers were used to amplify possible drug resistance genes, which encode different classes of major carbapenemases, including class A (Guiana extended-spectrum-lactamase (GES), imipenem-hydrolysing β-lactamase (IMI), Klebsiella pneumoniae carbapenemase (KPC), non-metallo carbapanemase (NMC), and Serratia Marcescens (SME)), class B (imipenemase (IMP), Verona integron-encoded metallo-β-lactamase (VIM), German imipenemase (GIM), Sao Paulo metallo-β-lactamases (SPM), Seoul imipenemase (SIM), and New Delhi metallo-β-lactamase (NDM)), and class D (oxacillinase (OXA)); ESBLs, including sulphydryl variable type β-lactamases (SHV), Temoniera (TEM), and cefotaxime-Munich (CTX-M); and AmpC β-lactamases, including Latamoxef (LAT), AmpC type (ACT), cephamycins (CMY), and Dhahran (DHA), to identify resistance genes of these strains [¹²–¹⁵]. Gene amplification was performed using Bio-Rad S1000 Thermal Cycler (Bio-Rad, Foster City, CA, USA). PCR products were analyzed by gel electrophoresis in 1.5% agarose and sequenced by the dye terminator method on the automatic sequencer ABI 3730 (Applied Biosystems, Carlsbad, CA, USA) after purification.

Nucleotide sequence analysis was performed by BLAST in the National Center for Biotechnology Information (NCBI) to identify β-lactamases genes.

Multilocus sequence typing (MLST)

Carbapenem-resistant K. pneumoniae (CRKP) isolates were grown overnight in Columbia blood plate medium at 37 °C, and DNA was extracted using the aforementioned method. Seven housekeeping genes, i.e., rpoB, gapA, mdh, pgi, phoE, infB, and tonB, were used to characterize CRKP isolates by MLST. Gene amplification and PCR product analysis were conducted using the same procedure as resistance gene analysis. Test parameters were according to those described in the site http://www.pasteur.fr/mlst/. Data were analyzed by eBURST version 3.0 with default settings.

Statistical analysis

Data were analyzed using SPSS version 11.02 (SPSS, Chicago, IL, USA). Categorical variables were expressed as numbers of strains (percentages) and compared by chi-square or Fisher’s exact test. A P value<0.05 was considered statistically significant.

Results

Strain characteristics

Of the 242 clinical CRE strains, 174 K. pneumoniae strains, 53 Escherichia coli strains, 12 Enterobacter cloacae strains, and one each for Enterobacter aerogenes, Serratia marcescens, and Citrobacter freundii strains were isolated, with corresponding isolation rates of 71.9%, 21.9%, 5.0%, 0.4%, 0.4%, and 0.4%, respectively. All clinical CRE strains were isolated from sputum, urine, blood, and secreta, with corresponding isolation rates of 55.0%, 26.0%, 10.3%, and 8.7%, respectively. Clinical CRE strains were also distributed in intensive care unit (41.3%), cardiovascular care unit and cadre’s ward (19.0%, respectively), nephrology department (7.4%), endocrinology department (3.7%), neurology and orthopedics departments (3.3%, respectively), burn department (2.1%), and dermatology outpatient department (0.8%). Patients with isolated strains were mostly long-term hospitalized patients (86.7%), and all of them were treated with carbapenem before, so combined use of amikacin, polymyxin and tigecycline was given. 147 cases were cured and 95 cases died.

Antimicrobial susceptibilities of CRE strains

Table 1 summarizes antibiotic susceptibilities of the 242 CRE strains observed in this study. High resistance rates (>70%) were observed against β-lactam antibiotics, ciprofloxacin, trimethoprim–sulfamethoxazole, and nitrofurantoin, among which resistance rates against β-lactam antibiotics accounted for more than 88.0%. Peak of resistance rate was observed against cefotaxime (99.17%). By contrast, resistance rates against tetracycline and aminoglycoside antibiotics were relatively low, i.e., between 8.3% and 62.4%. Among these antibiotics, resistance rate against tigecycline was the lowest (0.83%).

CNP test

In CNP test, 156 positive strains and 86 negative strains were detected. Among these strains, 156 CNP positive strains were all carbapenemase-positive strains, and one CNP negative strain was a K. pneumoniae strain carrying the bla_KPC-2 gene.

Distribution of β-lactamases and molecular characterization of bla genes in CRE strains

In this study, carbapenemases, ESBLs, and AmpC β-lactamase consisted mostly of β-lactamases produced by CRE strains (97.9%, 237/242). A total of 157 strains with carbapenemases, 185 strains with ESBLs, and 24 strains with AmpC β-lactamase were detected. These three types of β-lactamases coexisted in six strains. From 2010 to 2014, carbapenemase-carrying rates of these strains reached 56.4% (22/39), 51.4% (19/37), 61.7% (29/47), 72.4% (42/58), and 73.8% (45/61), and KPC-2 corresponded to the main type of carbapenemase detected in that time period. A strain carrying both bla_KPC-2 and bla_IMP-4 genes was detected in 2013.

Among all CRE strains, although one bla_SHV-1-carrying strain was detected, carrying rate of bla_SHV ESBL genes (bla_SHV-2, bla_SHV-5, bla_SHV-11, bla_SHV-12, and bla_SHV-142) was the highest (66.9%, 162/242), followed by those of bla_KPC class A carbapenemase genes (60.3%, 146/242) and other two genes, i.e., bla_TEM (46.3%, 112/242) and bla_CTX-M (26.0%, 63/242). Among these genes, bla_KPC was observed in most detected species, with a single genotype of bla_KPC-2. All related strains exhibited 100% similarity with each other and high homology with strains in China (NG_036335, 2008), Argentina (KM403446, 2014), and Portugal (JF323864, 2011). Carrying rate of bla_KPC-2 in Escherichia coli strains was higher than that in K. pneumoniae strains (77.4% versus 58.6%, P<0.001). However, inverse carrying rates were observed in bla_SHV (Escherichia coli versus K. pneumoniae, 5.6% versus 88.5%, P<0.001), bla_TEM (Escherichia coli versus K. pneumoniae, 3.8% versus 63.2%, P<0.001), and bla_CTX-M (Escherichia coli versus K. pneumoniae, 7.5% versus 33.3%, P<0.001).

bla_IMP-type class B carbapenemase genes were detected in 4.5% (11/242) of total CRE strains; these bla_IMP-type class B carbapenemase genes included bla_IMP-4 (6/11), bla_IMP-26 (3/11), and bla_IMP-6 (2/11). A single genotype was detected in one class B carbapenemase gene (bla_VIM-2) and in two AmpC β-lactamase genes (bla_ACT-1 and bla_CMY-2), with carrying rates of 0.4% (1/242), 6.6% (16/242), and 5.4% (13/242), respectively. Other β-lactamase genes, including bla_GES, bla_IMI, bla_NDM, bla_GIM, bla_SPM, bla_OXA, bla_LAT, and bla_DHA were not detected in the CRE strains.

MLST

A total of 174 CRKP were analyzed by MLST, and five ST types were detected. ST11 (91.2%) was the most frequent ST type, followed by ST23 (6.3%). ST15, ST1373, and ST1415 were detected in only one strain (0.6%). Results were compared with the K. pneumoniae MLST database in Zhejiang (http://www.pasteur.fr/mlst/) by eBURST version 3.0 (Fig. 1).

Discussion

As described previously, emergence of CRE became a rapidly growing global public health concern [¹⁶]. The China-CHINET surveillance in 2010 showed that the most frequent CRE in China was Klebsiella spp., particularly K. pneumonia [⁷]. Similarly, in this study, K. pneumonia was the most prevalent strain, followed by Escherichia coli, accounting for 71.9% and 21.9%, respectively. Thus, vigilance should be heightened when either of these two kinds of bacteria is isolated from clinical specimens. A total of 242 CRE strains were mainly retrieved from the intensive care unit, cardiovascular care unit, and cadre’s ward, in which CRE infection or colonization factors, including poor immune status, long hospital stay, and frequent use of antibiotics, were patient characteristics, indicating CRE reservoirs in the aforementioned departments. For this reason, hand hygiene and infection control measures must be emphasized. Monitoring of CRE must be strengthened to reduce CRE infection. Notably, two strains of CRE were isolated from outpatients. Although the proportion is small, the number of CRE strains is increasing.

Identical to data from China-CHINET (2010), CRE strains observed in this study were highly resistant to most common antimicrobial agents, except tigecycline, aminoglycoside, and tetracyclines. Currently, a number of susceptibility tests in vitro discovered that CRE strains are susceptible to polymyxin B, polymyxin E, tigecycline, and aminoglycosides, which were considered the top choice and last defense for treating CRE infections [¹⁷]. Therefore, the need to cautiously use such antimicrobial agents and distinguish colonized strains from pathogenic strains should be emphasized to avoid development of novel resistance. This study showed significant statistical differences in resistance rates of the two major prevalent species (K. pneumonia and Escherichia coli) against seven antimicrobial agents. Excluding tetracycline, resistance rates of K. pneumonia against the remaining six drugs, i.e., amikacin, tobramycin, nitrofurantoin, cefoxitin, cefepime, and ceftazidime, were higher than those of Escherichia coli. As mentioned previously, K. pneumonia was isolated mostly in all CRE strains, and its high resistance aggravated severity of drug resistance. Notably, CRE strains showed low resistance rate of against tigecycline, which belongs to glycylcycline antibiotics, resulting in advantages for future clinical treatment. However, application of tigecycline to blood and urinary tract infections should be discussed as a consequence of poor permeability in urinary tract and rapid distribution to body tissue after being released into the bloodstream [¹⁸]. Resistance rate of K. pneumonia against nitrofurantoin reached 92.5%; thus, clinical treatment should focus on timely prevention, isolation of suspected cases, and careful medication.

Data on genetic analysis performed on 242 CRE strains showed that 237 strains harbored bla genes. Notably, genetic characteristics of bla gene strains indicated that 123 strains harbored more than two kinds of β-lactamase genes, including carbapenemase, ESBLs, and AmpC genes. A CRE strain carrying carbapenemase genes was detected in 2013. Existence of these multiple bla gene strains (45.2%) indicated the complex resistance mechanisms that require further investigations.

Acquisition of genes encoding carbapenemases plays the most important role among the main mechanisms causing carbapenem resistance in Enterobacteriaceae [¹⁶]. In this study, the percentage of strains harboring carbapenemase genes reached 64.9% (157/242). Resistance mechanism of carbapenemase-negative producers to carbapenem possibly results from overexpression of ESBL and/or AmpC enzymes combined with outer-membrane porin loss, overexpression of efflux pump proteins to alter membrane permeability, or target alteration. Carrying rate of carbapenemase genes in CRE increased in the past five years. Among strains carrying carbapenemase genes, bla_KPC-2 yielded the highest detection rate, agreeing with its worldwide prevalence in recent years. Phylogenetic analysis also supported high identity among bla_KPC-2 strains in different countries. Although intra-transmission by clone is assignable, bla_KPC-2 gene, which is located in the plasmid, can be horizontally transferred by certain transposable components, such as plasmid and integron in Enterobacteriaceae, and can be used as a determinant because of its rapid and wide spread across the world [¹⁹,²⁰]. Thus, distribution of bla_KPC-2 in most species in this study hints a possible horizontal transmission rather than intraspecies dissemination. Phylogenetic analysis and homologous comparison with reference strains from NCBI identified six types of bla_SHV, including bla_SHV-1, bla_SHV-11, bla_SHV-12, bla_SHV-142, bla_SHV-2, and bla_SHV-5; two types of bla_TEM, including bla_TEM-1 and bla_TEM-135; and five types of bla_CTX-M, including bla_CTX-M-14, bla_CTX-M-65, bla_CTX-M-3, bla_CTX-M-15, and bla_CTX-M-55, in strains carrying resistant genes. bla_SHV was detected in approximately 88% of ESBL-producing Enterobacteriaceae, whereas bla_TEM and bla_CTX-M were present in three fifths and one thirds of the strains, respectively. These findings were concordant with studies published in China in recent years [²¹,²²]. bla_SHV-11, bla_TEM-1, and bla_CTX-M-14 were shown to be the predominant genotypes. In contrast to findings of this study, in European countries, Abdallah et al. reported that bla_CTX-M type was the most frequent β-lactamase encoding gene, and bla_CTX-M-15 was the dominant bla_CTX-M type, with a frequency of more than 90% [²³]. These geographic variations were reported previously [²⁴]. However, detailed reasons remain uncertain and require further investigation. β-lactamase genes were undetectable in the remaining five strains. Alteration of targets of carbapenem possibly involves this resistance mechanism.

Fig. 1 shows that ST11 was one of the ST types of K. pneumoniae in Zhejiang. Previous studies discovered that ST11 was the main clone that spreads bla_KPC and bla_NDM and can be related to resistance of carbapenem [²⁵–²⁸]. In this study, ST11 was the most frequent ST type in CRKP, indicating that ST11 clone must be prevented and controlled. ST15, ST23, ST1373, and ST1415 were only detected in this study. Fig. 1 shows that ST15 and ST352; ST23, ST1417, and ST1797; and ST1373, ST17, and ST1547 were detected in three separate clones. Fig. 1 also shows that ST1415 was observed in a single clone. These findings indicated variations in the strain.

In summary, in 2010–2014, occurrence of CRE in hospitals in Hangzhou, China was often accompanied by generation of multidrug-resistant genes, particularly bla_KPC-2 and bla_SHV-11. Tigecycline, aminoglycoside, and tetracyclines may be the last-line treatment for CRE. However, efficacy and toxicity are still subject to considerable public concern. CRKP was the most common CRE in this study, whereas ST11 was the most common ST type. Abuse of antimicrobial agents largely involves worldwide prevalence and increase in resistance rate of CRE. Efficiency of detection for CRE may also be affected by technological limitations. Therefore, effective prevention and control measures should be developed and strengthened to reduce the spread of CRE strains in hospitals and decrease their resistance rates.

References

[1] Papp-Wallace KM, Endimiani A, Taracila MA, Bonomo RA. Carbapenems: past, present, and future. Antimicrob Agents Chemother 2011; 55(11): 4943–4960

[2] Zhou T, Zhang X, Guo M, Ye J, Lu Y, Bao Q, Chi W. Phenotypic and molecular characteristics of carbapenem-non-susceptible Enterobacteriaceae from a teaching hospital in Wenzhou, southern China. Jpn J Infect Dis 2013; 66(2): 96–102

[3] Pollett S, Miller S, Hindler J, Uslan D, Carvalho M, Humphries RM. Phenotypic and molecular characteristics of carbapenem-resistant Enterobacteriaceae in a health care system in Los Angeles, California, from 2011 to 2013. J Clin Microbiol 2014; 52(11): 4003–4009

[4] Schwaber MJ, Lev B, Israeli A, Solter E, Smollan G, Rubinovitch B, Shalit I, Carmeli Y. Containment of a country-wide outbreak of carbapenem-resistant Klebsiella pneumoniae in Israeli hospitals via a nationally implemented intervention. Clin Infect Dis 2011; 52(7): 848–855

[5] El-Herte RI, Kanj SS, Matar GM, Araj GF. The threat of carbapenem-resistant Enterobacteriaceae in Lebanon: an update on the regional and local epidemiology. J Infect Public Health 2012; 5(3): 233–243

[6] Xu Y, Gu B, Huang M, Liu H, Xu T, Xia W, Wang T. Epidemiology of carbapenem resistant Enterobacteriaceae (CRE) during 2000–2012 in Asia. J Thorac Dis 2015; 7(3): 376–385

[7] Hu FP, Zhu DM, Wang F, Jiang XF, Yang Q, Huang WX, Jia B, Xu YH, Shen JL, Xu YC, Zhang XJ, Hu YJ, Ai XM, Zhang ZX, Ji P, Shan B, Du Y, Zhuo C, Su DH, Wang CQ, Wang AJ, Ni YX, Sun JY, Sun ZY, Chen ZJ, Wei LH, Wu L, Zhang H, Kong Q. CHINET surveillance of distribution and susceptibility of carbapenem-resistant Enterobacteriaceae isolates in 2010. Chin J Infect Chemother（Zhongguo Gan Ran Yu Hua Liao Za Zhi） 2013; 13(1): 1–7（in Chinese）

[8] Dai W, Sun S, Yang P, Huang S, Zhang X, Zhang L. Characterization of carbapenemases, extended spectrum β-lactamases and molecular epidemiology of carbapenem-non-susceptible Enterobacter cloacae in a Chinese hospital in Chongqing. Infect Genet Evol 2013; 14:1–7

[9] Rasmussen BA, Bush K, Keeney D, Yang Y, Hare R, O’Gara C, Medeiros AA. Characterization of IMI-1 β-lactamase, a class A carbapenem-hydrolyzing enzyme from Enterobacter cloacae. Antimicrob Agents Chemother 1996; 40(9): 2080–2086

[10] Clinical and Laboratory Standards Institute. Performance standards for antimiembial susceptibility testing; twenty-fourth informational supplement. CLSI document M100–S25. Wayne, PA: CLSI, 2015

[11] Zowawi HM, Sartor AL, Balkhy HH, Walsh TR, Al Johani SM, AlJindan RY, Alfaresi M, Ibrahim E, Al-Jardani A, Al-Abri S, Al Salman J, Dashti AA, Kutbi AH, Schlebusch S, Sidjabat HE, Paterson DL. Molecular characterization of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in the countries of the Gulf cooperation council: dominance of OXA-48 and NDM producers. Antimicrob Agents Chemother 2014; 58(6): 3085–3090

[12] Queenan AM, Bush K. Carbapenemases: the versatile β-lactamases. Clin Microbiol Rev 2007; 20(3): 440–458

[13] 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 β-lactamases in Enterobacteriaceae. J Antimicrob Chemother 2010; 65(3): 490–495

[14] Perez-Perez FJ, Hanson ND. Detection of plasmid-mediated AmpC β-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol 2002; 40(6): 2153–2162

[15] Nordmann P, Poirel L, Carrer A, Toleman MA, Walsh TR. How to detect NDM-1 producers. J Clin Microbiol 2011; 49(2): 718–721

[16] Nordmann P, Dortet L, Poirel L. Carbapenem resistance in Enterobacteriaceae: here is the storm! Trends Mol Med 2012; 18(5): 263–272

[17] Castanheira M, Sader HS, Deshpande LM, Fritsche TR, Jones RN. Antimicrobial activities of tigecycline and other broad-spectrum antimicrobials tested against serine carbapenemase- and metallo-β-lactamase-producing Enterobacteriaceae: report from the SENTRY Antimicrobial Surveillance Program. Antimicrob Agents Chemother 2008; 52(2): 570–573

[18] Kanj SS, Kanafani ZA. Current concepts in antimicrobial therapy against resistant Gram-negative organisms: extended-spectrum β-lactamase-producing Enterobacteriaceae, carbapenem-resistant Enterobacteriaceae, and multidrug-resistant Pseudomonas aeruginosa. Mayo Clin Proc 2011; 86(3): 250–259

[19] Stokes HW, Gillings MR. Gene flow, mobile genetic elements and the recruitment of antibiotic resistance genes into Gram-negative pathogens. FEMS Microbiol Rev 2011; 35(5): 790–819

[20] Partridge SR. Analysis of antibiotic resistance regions in Gram-negative bacteria. FEMS Microbiol Rev 2011; 35(5): 820–855

[21] Liu H, Wang Y, Wang G, Xing Q, Shao L, Dong X, Sai L, Liu Y, Ma L.The prevalence of Escherichia coli strains with extended spectrum beta-lactamases isolated in China. Front Microbiol 2015; 6:335

[22] An S, Chen J, Wang Z, Wang X, Yan X, Li J, Chen Y, Wang Q, Xu X, Li J, Yang J, Wang H, Gao Z. Predominant characteristics of CTX-M-producing Klebsiella pneumoniae isolates from patients with lower respiratory tract infection in multiple medical centers in China. FEMS Microbiol Lett 2012; 332(2): 137–145

[23] Abdallah HM, Wintermans BB, Reuland EA, Koek A, al Naiemi N, Ammar AM, Mohamed AA, Vandenbroucke-Grauls CM. Extended-spectrum β-lactamase- and carbapenemase-producing enterobacteriaceae isolated from Egyptian patients with suspected blood stream infection. PLoS One 2015; 10(5): e0128120

[24] Memariani M, Najar Peerayeh S, Zahraei Salehi T, Shokouhi Mostafavi SK. Occurrence of SHV, TEM and CTX-M β-lactamase genes among enteropathogenic Escherichia coli strains isolated from children with diarrhea. Jundishapur J Microbiol 2015; 8(4): e15620

[25] Giske CG, Fröding I, Hasan CM, Turlej-Rogacka A, Toleman M, Livermore D, Woodford N, Walsh TR. Diverse sequence types of Klebsiella pneumoniae contribute to the dissemination of blaNDM-1 in India, Sweden, and the United Kingdom. Antimicrob Agents Chemother 2012; 56(5): 2735–2738

[26] Pereira PS, de Araujo CFM, Seki LM, Zahner V, Carvalho-Assef APDA, Asensi MD. Update of the molecular epidemiology of KPC-2-producing Klebsiella pneumoniae in Brazil: spread of clonal complex 11 (ST11, ST437 and ST340). J Antimicrob Chemother 2013; 68(2): 312–316

[27] Chen L, Mathema B, Chavda KD, DeLeo FR, Bonomo RA, Kreiswirth BN. Carbapenemase-producing Klebsiella pneumoniae: molecular and genetic decoding. Trends Microbiol 2014; 22(12): 686–696

[28] Kim SY, Shin J, Shin SY, Ko KS. Characteristics of carbapenem-resistant Enterobacteriaceae isolates from Korea. Diagn Microbiol Infect Dis 2013; 76(4): 486–490

RIGHTS & PERMISSIONS

Higher Education Press and Springer-Verlag Berlin Heidelberg