Emergence of carbapenem-resistant clinical Enterobacteriaceae isolates from a teaching hospital in Shanghai, China

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1 Journal of Medical Microbiology (2012), 61, DOI /jmm Emergence of carbapenem-resistant clinical Enterobacteriaceae isolates from a teaching hospital in Shanghai, China Fupin Hu, 1 3 Shudan Chen, 1,2 3 Xiaogang Xu, 1 Yan Guo, 1 Yang Liu, 1 Demei Zhu 1 and Yingyuan Zhang 1 Correspondence Demei Zhu zhu_dm@fudan.edn.cn 1 Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai , PR China 2 State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai , PR China Received 18 July 2011 Accepted 6 September 2011 Carbapenems such as imipenem and meropenem are first-line agents for the treatment of serious nosocomial infections caused by multidrug-resistant clinical isolates of bacteria belonging to the family Enterobacteriaceae. However, resistance to carbapenems has increased dramatically among members of the family Enterobacteriaceae isolated from a teaching hospital in Shanghai, China. In the present study, we investigated the prevalence and molecular characteristics of carbapenemresistant clinical isolates of Enterobacteriaceae. None of the 77 clinical isolates collected from 2002 to 2009 were susceptible to ertapenem and only 6.5 % and 1.3 % of isolates were susceptible to imipenem and meropenem, respectively. Colistin and tigecycline were found to be the most active agents against carbapenem-resistant Enterobacteriaceae isolates, inhibiting 90 % of isolates at a concentration of 1 mg ml 1 and 4 mg ml 1, respectively. The results of PFGE analysis suggested that many of the KPC-2-producing isolates of Citrobacter freundii and Klebsiella pneumoniae were clonally related. Most of these isolates were isolated from the same ward, namely the neurosurgical ward, suggesting horizontal transfer of the KPC-2-encoding gene in these isolates. Of the 77 isolates, 84.4 % were found, by PCR, to be capable of carbapenemase production. SDS-PAGE analysis revealed that 75.3 % (58/77) of the isolates had lost at least one porin protein. Our results suggested that the prompt detection of carbapenemase-producing strains is critical for the containment of nosocomial transmission. As no novel antimicrobials have been identified for use in the treatment of these pan-drug-resistant isolates, further studies should focus on the rational use of available antibiotics, the implementation of active antibiotic resistance surveillance and the strict implementation of infection control measures to avoid the rapid spread or outbreak of carbapenemase-producing Enterobacteriaceae in health-care facilities. INTRODUCTION Carbapenems such as imipenem and meropenem are firstline agents for the treatment of serious healthcare-associated infections caused by clinical multidrug-resistant Enterobacteriaceae (Brink et al., 2004).However,the phenomenon of carbapenem resistance is emerging in a wide variety of these species (Yigit et al., 2001). Carbapenemases have been widely detected in recent years and predominantly contribute to carbapenem resistance among members of the family Enterobacteriaceae including Klebsiella pneumoniae and Citrobacter freundii (Peleg & Hooper, 2010). Carbapenemases including KPC, VIM, IPM, SME and OXA belong to various families of b-lactamases (Queenan & Bush, 2007). Infections 3These authors contributed equally to this work. Abbreviations: CRE, carbapenem-resistant Enterobacteriaceae; ESBL, extended-spectrum b-lactamase; MBL, metallo-b-lactamase. caused by carbapenem-resistant Enterobacteriaceae (CRE) have presented a great challenge to physicians in recent years (CDC, 2009; Munoz-Price & Quinn, 2009). In Shanghai, China, K. pneumoniae is the second most frequently isolated pathogen among Gram-negative bacilli according to the Shanghai Bacterial Resistance Surveillance Program (Zhu et al., 2006, 2007). Although the prevalence of carbapenemresistant isolates among the Enterobacteriaceae is low, it has increased dramatically in recent years. In this study, CRE clinical isolates were collected and analysed for their molecular characteristics and similarities. METHODS Bacterial strains. All Enterobacteriaceae isolates obtained between January 2002 and April 2009 (n57775) from Huashan Hospital, Fudan University, Shanghai, China, a 1300-bed tertiary care hospital, G 2012 SGM Printed in Great Britain

2 Emergence of carbapenem-resistant Enterobacteriaceae were screened to investigate trends in the prevalence of the CRE isolates. These isolates were non-duplicate and were collected on routine workdays without any specific exclusion criteria. From the 7775 isolates, a total of 77 were selected for testing, including 43 isolates of K. pneumoniae, one isolate of Klebsiella oxytoca, four isolates of Enterobacter cloacae, one isolate of Escherichia coli, one isolate of Serratia marcescens, one isolate of Providencia rettgeri, one isolate of Citrobacter farmeri and 25 isolates of C. freundii, most of which were resistant to all of three carbapenems tested, namely imipenem, meropenem and ertapenem, and had been isolated from different patients. Of the 77 isolates, 57.1 % (44/77) were isolated from urine, 39.0 % (30/77) from sputum, 1.3 % (1/77) from negativepressure ball drainage, 1.3 % (1/77) from blood and 1.3 % (1/77) from cerebrospinal fluid. All of the strains were isolated from hospitalized patients. A well-characterized strain of C. freundii, producing an IMP- 9-type metallo-b-lactamase (MBL), and E. coli strain ATCC were used as positive and negative controls, respectively, for antimicrobial susceptibility testing. Antimicrobial susceptibility testing. Antimicrobial susceptibility testing was performed using the disc diffusion method and the agar dilution method recommended by the Clinical and Laboratory Standards Institute (CLSI, 2010). MICs of amikacin, cefotaxime, ceftazidime, cefepime, aztreonam, ertapenem, imipenem, meropenem, doxycycline, minocycline and ciprofloxacin were determined following the criteria of the CLSI. Breakpoint MICs of tigecycline were determined following the guidelines of the US Food and Drug Administration (MICs 2 mgl 21 denoting susceptibility and 8 mgl 21 denoting resistance). MICs of colistin were interpreted following the criteria of the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2011) (MICs 2 mgl 21 denoting susceptibility and 4 mgl 21 denoting resistance). Genotypic detection of b-lactamase genes. The presence of genes encoding the b-lactamases TEM, SHV, PER, SFO, VEB, CTX-M and GES, and genes encoding the plasmid-borne AmpCs and carbapenemases NDM-1, VIM, NDM-1, IPM, SPM, SME, GIM, NMC, IMI, IND and OXA was investigated in all of the 77 clinical strains by using previously described primers (Chen et al., 2011; Murray et al., 2007). Two conserved primers, forward primer KPC-F (59-AGGACTTTG- GCGGCTCCAT-39) and reverse primer KPC-R (59-TCCCTCGAG- CGCGAGTCTA-39), were designed against the bla KPC gene to detect the sequences encoding KPC-1 to KPC-11, according to the published nucleotide sequences in GenBank. PCR amplicons were sequenced and the DNA sequences obtained were compared to those available in the NCBI GenBank database using BLAST searches. Epidemiological studies. Isolates of K. pneumoniae and C. freundii were typed by using PFGE, which was performed according to a previously described protocol (Hunter et al., 2005). The DNA fingerprints generated were analysed according to the criteria proposed by Tenover et al. (1995). Outer-membrane proteins. SDS-PAGE analysis of outer-membrane proteins, including OmpK35, OmpK36 and OmpK37, of all CRE isolates was performed as described previously (Woodford et al., 2004; Hosaka et al., 1995). RESULTS Antimicrobial susceptibility testing and prevalence of CRE isolates The prevalence of CRE clinical isolates increased significantly in 2009, especially for K. pneumoniae (Fig. 1). The detection rate of carbapenem-resistant isolates of K. pneumoniae rose from 0.91 % (5/549) in 2005 to % (82/637) in The proportion of imipenem-resistant isolates of C. freundii rose rapidly during , from % (3/27) to % (9/22), and remained high at 33.3 % (6/18) in Of the 77 clinical isolates selected for this study, none was found to be susceptible to ertapenem, cefotaxime, aztreonam or piperacillin tazobactam. The proportions of the isolates that were susceptible to imipenem, meropenem, colistin and tigecycline were 6.5 %, 1.3 %, 96.1 % and 88.3 %, respectively, and.70 % of the isolates were susceptible to minocycline and doxycycline (Table 1). Characterization of b-lactamases in CRE isolates PCR was used to detect genes encoding NDM-1, VIM-2, SPM, GIM, NMC, IMI, IND, SME, OXA-48, OXA-50, OXA-55, OXA-60, OXA-69 and OXA-24 carbapenemases but did not result in amplicons with any of the clinical isolates. Of the 77 isolates, 65 (84.4 %) were found to be carbapenemase-producing. The bla KPC gene was detected in 58/77 (75.3 %) isolates and in all cases the gene encoded KPC-2-type carbapenemase. Among these 58 strains, this Fig. 1. Prevelance of CRE clinical isolates from 2002 to 2009 in Shanghai, China.

3 F. Hu and others Table 1. Activities of various antimicrobial agents against 77 CRE isolates R, Resistant; S, susceptible. All organisms were deemed resistant or susceptible based on the criteria of the CLSI, unless indicated otherwise. Drug MIC range (mg ml 1 ) MIC 50 (mg ml 1 ) MIC 90 (mg ml 1 ) R (%) S (%) Imipenem Meropenem Ertapenem Colistin D 96.1D Tigecycline * 88.3* Minocycline Doxycycline Cefepime Ceftazidime Cefotaxime Cefoxitin Cefoperazone/sulbactam Piperacillin tazobactam Aztreonam Amikacin Ciprofloxacin *Determined following the guidelines of the US Food and Drug Administration. DInterpreted following the criteria of EUCAST (2011). gene was coupled with the bla VIM-1 gene in two isolates, the bla IMP-2 gene in two isolates and the bla IMP-1 gene in one isolate. The genes encoding VIM-1, IMP-1 and IMP-2 carbapenemases were also detected in five (6.5 %), four (5.2 %) and three (3.9 %) isolates, respectively. The gene encoding CTX-M b-lactamase was detected in 65/77 (84.4 %) isolates, of which one possessed the bla CTX-M-2 gene, 55 possessed the bla CTX-M-14 gene and 20 possessed the bla CTX-M-15 gene. Plasmid-borne AmpCs DHA-1 or CMY-2 were detected in 31/77 (40.3 %) isolates. Table 2. PFGE analysis of carbapenem-resistant isolates of C. freundii and K. pneumoniae Values represent number of isolates with the same banding pattern followed, in parentheses, by the proportion of the total number of isolates as a percentage. Banding pattern Number (%) of isolates K. pneumoniae C. freundii A 5 (11.6) 2 (8.0) B 4 (9.3) 1 (4.0) C 5 (11.6) 7 (28.0) D 4 (9.3) 15 (60.0) E 6 (14.0) F 6 (14.0) G 2 (4.7) H 2 (4.7) I 2 (4.7) J 7 (16.3) Total PFGE and analysis of outer-membrane proteins The results of PFGE analysis, interpreted according to Tenover et al. (1995), revealed four and ten unrelated genotypes in isolates of C. freundii and K. pneumoniae, respectively (Table 2). SDS-PAGE profiles of outer-membrane proteins, including OmpK35, OmpK36 and OmpK37, indicated that 75.3 % (58/77) of the isolates possessed, among others, a single band probably corresponding to OmpK35 or OmpK36, suggesting that they have lost at least one porin protein (see Fig. 2). DISCUSSION The first CRE isolate (E. coli) collected from Huashan training hospital was recovered from the urine of patients in a medical ward in March 2002, later followed by the isolation of carbapenem-resistant strains of C. freundii and K. pneumoniae. In 2009, the incidence of imipenemresistant Enterobacteriaceae rose rapidly. These results suggested that the increased prevalence of carbapenemresistant isolates in the hospital may have been caused by a failure to control the spread of these strains. As CRE are some of the most important Gram-negative bacilli residing in hospitals and causing hospital-acquired infections, the emergence of CRE isolates has attracted much attention (Schwaber & Carmeli, 2008). Although carbapenemase-producing Enterobacteriaceae isolates are usually pan-drug resistant, several isolates may still remain susceptible to amikacin and ciprofloxacin. In the present study, 10.4 % and 13.0 % of isolates were found to be susceptible to amikacin and ciprofloxacin, respectively. 134 Journal of Medical Microbiology 61

4 Emergence of carbapenem-resistant Enterobacteriaceae Fig. 2. SDS-PAGE analysis of outer-membrane proteins of K. pneumoniae clinical isolates (partial results). Lanes: M, marker; 1 and 6, carbapenem-susceptible K. pneumoniae isolates from different patients showing two bands representing porins OmpK35 (40 kda) and OmpK36 (37 kda) (marked a and b, respectively); 2 5, carbapenem-resistant K. pneumoniae isolates from different patients. Many studies have demonstrated the activity of colistin and tigecycline against carbapenemase-producing Enterobacteriaceae isolates (Pliatsika et al., 2007; Bratu et al., 2005). Colistin was found to be most active in our study, inhibiting 90 % of isolates at a concentration of 1 mg ml 21 (MIC 90 ). Of the nine tigecycline-resistant isolates, eight were found to be susceptible to colistin. However, the potential toxicity of colistin and the need for it to be used with other antimicrobial agents narrows its potential for clinical use (Walsh et al., 2005). In the present study, ~70 % of carbapenem-resistant isolates were susceptible to both minocycline and doxycycline and ~89 % were susceptible to tigecycline. Therefore, we presume that these drugs would be a rational option for the treatment of severe infections caused by CRE isolates. Although the production of extended-spectrum b-lactamases (ESBLs) and/or plasmid-borne AmpC b-lactamases, coupled with porin protein loss, can be responsible for carbapenem resistance (Jacoby et al., 2004; Girlich et al., 2009; Carvalhaes et al., 2010), the main agents of resistance to carbapenems are carbapenemases, especially KPC and MBLs, such as VIM and IPM (Hirsch & Tam, 2010). The genes encoding carbapenemases are usually carried by plasmids that often carry other resistance genes simultaneously, resulting in extensively drug-resistant bacteria (Gootz et al., 2009). In this study, out of the 25 C. freundii isolates, 18 carried KPC-2 carbapenemase genes and five produced both KPC-2 carbapenemase and MBL as well as CTX-M-14-type ESBL and CMY-2-type plasmid-borne AmpC. Out of the 43 K. pneumoniae isolates, nine carried carbapenemase genes encoding CTX-M-14-type ESBL and DHA-1-type plasmid-borne AmpC. In these two species, 23.3 % (10/43) of K. pneumoniae isolates and 24 % (6/25) of C. freundii isolates carried more than two kinds of carbapenemase genes. Although widespread dissemination of NDM-producing Enterobacteriaceae has been reported in India, Pakistan, the UK and other countries (Kumarasamy et al., 2010; Rolain et al., 2010), China still has no reports of NDM-producing Enterobacteriaceae, although there have been reports of NDM-producing strains of Acinetobacter baumannii (Chen et al., 2011). In this study, we screened 77 isolates of Enterobacteriaceae for the bla NDM-1 gene; however, none was found to be NDM-1-producing. Since plasmids are readily transferable, carbapenem-resistance genes can easily spread within species and even from species to species, such as C. freundii and K. oxytoca (Schwaber & Carmeli, 2008; Sidjabat et al., 2009). When such plasmids enter a rapidly disseminating bacterial strain, the result may be a widespread outbreak of a multi-drugresistant pathogen (Schwaber & Carmeli, 2008). The PFGE profiles suggested that many of the KPC-2-producing isolates of C. freundii and K. pneumoniae were clonally related and most of these isolates were isolated from the same ward, namely the neurosurgical ward. Of the 25 C. freundii isolates, 15 possessed the same genotype and among the 43 K. pneumoniae isolates, 12 displayed the same PFGE pattern, suggesting that horizontal spread of the bla KPC-2 gene occurs in C. freundii and K. pneumoniae. In contrast, the four isolates of E. cloacae showed four different banding patterns. The emergence of conferred carbapenemase resistance in strains in a hospital in China is alarming because it adds to the already difficult task of treating antimicrobial-resistant infections. Because carbapenemase-resistance genes can potentially spread rapidly via transferable plasmids, infections caused by carbapenemase-producing Enterobacteriaceae may prove difficult to control once they emerge (Pournaras et al., 2009), especially for both KPC- and MBL-producing isolates. Therefore, prompt detection is critical for the containment of carbapenemase-producing strains and for the prevention of nosocomial transmission (Hirsch & Tam, 2010; Lee et al., 2003; Pournaras et al., 2010).Asveryfew novel antimicrobials have been discovered for the treatment of these pan-drug-resistant isolates, further studies should focus on the rational use of available antibiotics, implementation of active antibiotic resistance surveillance and implementation of strict infection control measures to avoid the rapid spread or outbreak of carbapenemase-producing Enterobacteriaceae in health-care facilities. ACKNOWLEDGEMENTS We kindly thank Thomas K. W. Ling from the Department of Microbiology, The Chinese University of Hong Kong (Prince of Wales Hospital, Hong Kong, Special Administrative Region, People s Republic of China), for critical reading of this manuscript. This work was supported by the Shanghai Municipal Natural Science Foundation (11ZR ). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript

5 F. Hu and others REFERENCES Bratu, S., Tolaney, P., Karumudi, U., Quale, J., Mooty, M., Nichani, S. & Landman, D. (2005). Carbapenemase-producing Klebsiella pneumoniae in Brooklyn, NY: molecular epidemiology and in vitro activity of polymyxin B and other agents. J Antimicrob Chemother 56, Brink, A. J., Feldman, C., Grolman, D. C., Muckart, D., Pretorius, J., Richards, G. A., Senekal, M. & Sieling, W. (2004). Appropriate use of the carbapenems. S Afr Med J 94, Carvalhaes, C. G., Picão, R. C., Nicoletti, A. G., Xavier, D. E. & Gales, A. C. (2010). Cloverleaf test (modified Hodge test) for detecting carbapenemase production in Klebsiella pneumoniae: be aware of false positive results. J Antimicrob Chemother 65, CDC (2009). Guidance for control of infections with carbapenemresistant or carbapenemase-producing Enterobacteriaceae in acute care facilities. MMWR Morb Mortal Wkly Rep 58, Chen, Y., Zhou, Z., Jiang, Y. & Yu, Y. (2011). Emergence of NDM-1- producing Acinetobacter baumannii in China. J Antimicrob Chemother 66, CLSI (2010). Performance Standards for Antimicrobial Susceptibility Testing: Twentieth Informational Supplement. M100-S20. Wayne, PA: Clinical and Laboratory Standards Institute. EUCAST (2011). Breakpoint tables for interpretation of MICs and zone diameters (version 1.3, January 5, 2011). fileadmin/src/media/pdfs/eucast_files/disk_test_documents/eucast _breakpoints_v1.3_pdf.pdf Girlich, D., Poirel, L. & Nordmann, P. (2009). CTX-M expression and selection of ertapenem resistance in Klebsiella pneumoniae and Escherichia coli. Antimicrob Agents Chemother 53, Gootz, T. D., Lescoe, M. K., Dib-Hajj, F., Dougherty, B. A., He, W., Della-Latta, P. & Huard, R. C. (2009). Genetic organization of transposase regions surrounding bla KPC carbapenemase genes on plasmids from Klebsiella strains isolated in a New York City hospital. Antimicrob Agents Chemother 53, Hirsch, E. B. & Tam, V. H. (2010). Detection and treatment options for Klebsiella pneumoniae carbapenemases (KPCs): an emerging cause of multidrug-resistant infection. J Antimicrob Chemother 65, Hosaka, M., Gotoh, N. & Nishino, T. (1995). Purification of a 54- kilodalton protein (OprJ) produced in NfxB mutants of Pseudomonas aeruginosa and production of a monoclonal antibody specific to OprJ. Antimicrob Agents Chemother 39, Hunter, S. B., Vauterin, P., Lambert-Fair, M. A., Van Duyne, M. S., Kubota, K., Graves, L., Wrigley, D., Barrett, T. & Ribot, E. (2005). Establishment of a universal size standard strain for use with the PulseNet standardized pulsed-field gel electrophoresis protocols: converting the national databases to the new size standard. J Clin Microbiol 43, Jacoby, G. A., Mills, D. M. & Chow, N. (2004). Role of b-lactamases and porins in resistance to ertapenem and other b-lactams in Klebsiella pneumoniae. Antimicrob Agents Chemother 48, Kumarasamy, K. K., Toleman, M. A., Walsh, T. R., Bagaria, J., Butt, F., Balakrishnan, R., Chaudhary, U., Doumith, M., Giske, C. G. & other authors (2010). Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Lancet Infect Dis 10, Lee, K., Lim, Y. S., Yong, D., Yum, J. H. & Chong, Y. (2003). Evaluation of the Hodge test and the imipenem-edta double-disk synergy test for differentiating metallo-b-lactamase-producing isolates of Pseudomonas spp. and Acinetobacter spp. J Clin Microbiol 41, Munoz-Price, L. S. & Quinn, J. P. (2009). The spread of Klebsiella pneumoniae carbapenemases: a tale of strains, plasmids, and transposons. Clin Infect Dis 49, Murray, P. R., Baron, E. J., Jorgensen, J. H., Landry, M. L. & Pfaller, M. A. (2007). Detection and characterization of antimicrobial resistance genes in pathogenic bacteria. In Manual of Clinical Microbiology, 9th edn, pp Edited by J. K. Rasheed, F. Cockerill & F. C. Tenover. Washington, DC: American Society for Microbiology. Peleg, A. Y. & Hooper, D. C. (2010). Hospital-acquired infections due to Gram-negative bacteria. N Engl J Med 362, Pliatsika, V., Afkou, Z., Protonotariou, E. & Sofianou, D. (2007). In vitro activity of tigecycline against metallo-b-lactamase-producing Enterobacteriaceae. J Antimicrob Chemother 60, Pournaras, S., Protonotariou, E., Voulgari, E., Kristo, I., Dimitroulia, E., Vitti, D., Tsalidou, M., Maniatis, A. N., Tsakris, A. & Sofianou, D. (2009). Clonal spread of KPC-2 carbapenemase-producing Klebsiella pneumoniae strains in Greece. J Antimicrob Chemother 64, Pournaras, S., Poulou, A. & Tsakris, A. (2010). Inhibitor-based methods for the detection of KPC carbapenemase-producing Enterobacteriaceae in clinical practice by using boronic acid compounds. J Antimicrob Chemother 65, Queenan, A. M. & Bush, K. (2007). Carbapenemases: the versatile b- lactamases. Clin Microbiol Rev 20, Rolain, J. M., Parola, P. & Cornaglia, G. (2010). New Delhi metallo-blactamase (NDM-1): towards a new pandemia? Clin Microbiol Infect 16, Schwaber, M. J. & Carmeli, Y. (2008). Carbapenem-resistant Enterobacteriaceae: a potential threat. JAMA 300, Sidjabat, H. E., Silveira, F. P., Potoski, B. A., Abu-Elmagd, K. M., Adams-Haduch, J. M., Paterson, D. L. & Doi, Y. (2009). Interspecies spread of Klebsiella pneumoniae carbapenemase gene in a single patient. Clin Infect Dis 49, Tenover, F. C., Arbeit, R. D., Goering, R. V., Mickelsen, P. A., Murray, B. E., Persing, D. H. & Swaminathan, B. (1995). Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 33, Walsh, T. R., Toleman, M. A., Poirel, L. & Nordmann, P. (2005). Metallo-b-lactamases: the quiet before the storm? Clin Microbiol Rev 18, Woodford, N., Tierno, P. M., Jr, Young, K., Tysall, L., Palepou, M. F. I., Ward, E., Painter, R. E., Suber, D. F., Shungu, D. & other authors (2004). Outbreak of Klebsiella pneumoniae producing a new carbapenem-hydrolyzing class A b-lactamase, KPC-3, in a New York Medical Center. Antimicrob Agents Chemother 48, Yigit, H., Queenan, A. M., Anderson, G. J., Domenech-Sanchez, A., Biddle, J. W., Steward, C. D., Alberti, S., Bush, K. & Tenover, F. C. (2001). Novel carbapenem-hydrolyzing b-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae. Antimicrob Agents Chemother 45, Zhu, D., Zhang, Y. & Wang, F. (2006). Surveillance of bacterial resistance in Shanghai hospitals during Chin J Infect Chemother 7, Zhu, D., Zhang, Y. & Wang, F. (2007). Surveillance of bacterial resistance from hospitals in Shanghai in Chin J Infect Chemother 8, Journal of Medical Microbiology 61