Demei Zhu

vanM, a New Glycopeptide Resistance Gene Cluster Found in Enterococcus faecium

ABSTRACT Since glycopeptide-resistant enterococci (GRE) were reported in 1988, they have appeared in hospitals worldwide. Seven van gene cluster types (vanA, vanB, vanC, vanD, vanE, vanG, and vanL) are currently known. We investigated a clinical strain of Enterococcusfaecium Efm-HS0661 that was isolated in 2006 from an inpatient with intra-abdominal infection in Shanghai. It was resistant to most antimicrobials, including vancomycin (MIC, >256 μg/ml) and teicoplanin (MIC, 96 μg/ml). Glycopeptide resistance could be transferred to E. faecium BM4105RF by conjugation. The donor and its transconjugant were negative by PCR for the known van genes. By cloning and primer walk sequencing, we discovered a novel van gene cluster, designated vanM. The vanM ligase gene was 1,032-bp in length and encoded a 343-amino-acid protein that shared 79.9, 70.8, 66.3, and 78.8% amino acid identity with VanA, VanB, VanD, and VanF, respectively. Although the vanM DNA sequence was closest to vanA, the organization of the vanM gene cluster was most similar to that of vanD. Upstream from the vanM cluster was an IS1216-like element, which may play a role in the dissemination of this resistance determinant. Liquid chromatography-mass spectrometry analysis of peptidoglycan precursors extracted from the VanM-type strain Efm-HS0661 treated with vancomycin or teicoplanin revealed a modified precursor (UDP-N-acetylmuramic acid [MurNAc]-tetrapeptide-d-Lac), indicating that VanM, like VanA, confers glycopeptide resistance by the inducible synthesis of precursor ending in d-Ala-d-Lac.

Antimicrobial Susceptibility of Mycoplasma pneumoniae Isolates and Molecular Analysis of Macrolide-Resistant Strains from Shanghai, China

ABSTRACT Fifty-three Mycoplasma pneumoniae strains were isolated from pediatric patients in Shanghai, China, from October 2005 to February 2008. Of 53 clinical isolates, 44 (83%) were resistant to erythromycin (MICs of >128 μg/ml for all 44 strains), azithromycin, and clarithromycin. All macrolide-resistant M. pneumoniae strains harbored an A-to-G transition mutation at position 2063 in 23S rRNA genes. Forty-five (85%) clinical isolates were classified into the P1 gene restriction fragment length polymorphism type I, and six (11%) were type II.

Characterization of macrolide resistance in Mycoplasma pneumoniae isolated from children in Shanghai, China

One hundred Mycoplasma pneumoniae strains were isolated from pediatric patients from March 2008 to July 2009. Of 100 isolates, 90 (90%) were resistant to erythromycin (MICs >128 microg/mL for 88 strains and 64 microg/mL for 2 strains), azithromycin, and clarithromycin. Fluoroquinolones and tetracyclines maintain good activities against clinical M. pneumoniae isolates. Of 90 macrolide-resistant M. pneumoniae strains, 88 (98%) harbored an A-to-G transition mutation at position 2063 in 23S rRNA genes, and the remaining 2 showed either A2064G or A2063T mutation; the latter point mutation is newly discovered and reported. Ninety-three (93%) clinical isolates were classified into the P1 gene restriction fragment length polymorphism (RFLP) type I, and 7 (7%) were type II.

Investigation of extended-spectrum β-lactamase in Klebsiellae pneumoniae and Escherichia coli from China

Extended-spectrum beta-lactamases (ESBLs) are an increasing cause of resistance to third-generation cephalosporins in Enterobacteriaceae. However, they have not been well studied in China. We investigated the prevalence, resistance, and probable gene type of ESBLs using MICs testing and polymerase chain reaction in 559 Klebsiellae pneumoniae and 427 Escherichia coli isolates collected from patients in Huashan Hospital from 1 January to 31 December 1999. The incidence of ESBL-producing strains was 51% among Klebsiellae pneumoniae (285/559) and 23.6% among Escherichia coli (101/427), most of which were collected from patients in intensive care unit and neurosurgical ward. PFGE showed that some epidemic ESBL-producing strains were present in the ICU, especially among ESBL-producing Klebsiellae pneumoniae. The major source of ESBL-producing Klebsiellae pneumoniae and Escherichia coli was sputum specimen (63.5%) and urine (64.3%), respectively. These strains were resistant to most beta-lactams (including the third-generation cephalosporins and monobactams) and non-beta-lactams (such as fluoroquinolones, aminoglycosides, tetracycline, and chloramphenicol), all or most ESBL producers were susceptible to imipenem, cefmetazole and beta-lactam/clavulanic acid. TEM was the main type of beta-lactamases and the CTX-M type of ESBLs was common in these isolates. Some ESBL-producing Escherichia coli and most ESBL-producing Klebsiellae pneumoniae produced more than one type of beta-lactamase. These data confirm that ESBL producers are common among hospital strains of Escherichia coli and Klebsiellae pneumoniae in China. It is important to monitor such strains closely and prevent their spread.

Susceptibility of Extended-Spectrum-β-Lactamase-Producing Enterobacteriaceae According to the New CLSI Breakpoints

ABSTRACT In 2010 the Clinical and Laboratory Standards Institute (CLSI) lowered the susceptibility breakpoints of some cephalosporins and aztreonam for Enterobacteriaceae and eliminated the need to perform screening for extended-spectrum β-lactamases (ESBLs) and confirmatory tests. The aim of this study was to determine how many ESBL-producing strains of three common species of Enterobacteriaceae test susceptible using the new breakpoints. As determined with the CLSI screening and confirmatory tests, 382 consecutive ESBL-producing strains were collected at Huashan Hospital between 2007 and 2008, including 158 strains of Escherichia coli, 164 of Klebsiella pneumoniae, and 60 of Proteus mirabilis. Susceptibility was determined by the CLSI agar dilution method. CTX-M-, TEM-, and SHV-specific genes were determined by PCR amplification and sequencing. bla CTX-M genes alone or in combination with bla SHV were present in 92.7% (354/382) of these ESBL-producing strains. Forty-two (25.6%) strains of K. pneumoniae harbored SHV-type ESBLs alone or in combination. No TEM ESBLs were found. Utilizing the new breakpoints, all 382 strains were resistant to cefazolin, cefotaxime, and ceftriaxone, while 85.0 to 96.7% of P. mirabilis strains tested susceptible to ceftazidime, cefepime, and aztreonam, 41.8 to 45.6% of E. coli strains appeared to be susceptible to ceftazidime and cefepime, and 20.1% of K. pneumoniae were susceptible to cefepime. In conclusion, all ESBL-producing strains of Enterobacteriaceae would be reported to be resistant to cefazolin, cefotaxime, and ceftriaxone by using the new CLSI breakpoints, but a substantial number of ESBL-containing P. mirabilis and E. coli strains would be reported to be susceptible to ceftazidime, cefepime, and aztreonam, which is likely due to the high prevalence of CTX-M type ESBLs.

High Prevalence of KPC-2 type Carbapenemase Coupled with CTX-M type Extended-Spectrum β-Lactamases in Carbapenem-Resistant Klebsiella pneumoniae in a Teaching Hospital in China

Carbapenems such as imipenem and meropenem are the first-line agents for the treatment of serious nosocomial infections caused by multidrug-resistant Enterobacteriaceae clinical isolates (2). However, the phenomenon of carbapenem resistance is emerging among a wide variety of these species (11). Carbapenemases have been widespread in recent years and predominantly contribute to carbapenem resistance among Enterobacteriaceae, especially Klebsiella pneumoniae and Citrobacter freundi (8). For screening of the carbapenem-resistant Enterobacteriaceae clinical isolates, we conducted a surveillance study of carbapenem-resistant Enterobacteriaceae isolates in order to investigate the frequency and prevalence of K. pneumoniae carbapenemase (KPC)-type genes in Huashan Hospital (Fudan University, Shanghai, China), a 1,300-bed tertiary care hospital. We hereby report a high prevalence of carbapenem-resistant K. pneumoniae isolates carrying the KPC-2-type carbapenemase gene or KPC-2-type carbapenemase gene coupled with the CTX-M-type extended-spectrum β-lactamase (ESBL) gene. From January 2005 to March 2010, 109 nonduplicate Klebsiella pneumoniae isolates with resistance to ertapenem were collected. Antimicrobial susceptibility testing was performed by the agar dilution method, and MICs were interpreted following CLSI (4) or British Society for Antimicrobial Chemotherapy (BSAC) criteria (1). The presence of genes encoding β-lactamases, including metallo-β-lactamases (MBLs), KPC-type and OXA-type carbapenemases, ESBLs, and plasmid-mediated AmpC enzymes were screened by PCR using primers described by Rasheed et al. (10). All amplified products were then subjected to direct nucleotide sequencing. The results were analyzed with the software available on the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov). The susceptibility rates of 109 K. pneumoniae isolates to imipenem, meropenem, and ertapenem were 4.6%, 2.8%, and 0.0%, respectively (Table 1). Carbapenem-resistant Enterobacteriaceae isolates exhibited high resistance rates against the carbapenem agents tested, with MIC90 values of 128 μg/ml or higher. Totals of 96.3%, 74.3%, 66.1%, and 64.2% of isolates were susceptible to colistin, minocycline, tigecycline, and doxycycline, respectively (Table 1). KPC-2-type carbapenemase was the most predominant carbapenemase, present in 70.6% (77/109) of isolates. KPC-2-type carbapenemase coupled with CTX-M-14- or CTX-M-15-type ESBL accounted for 59.6% (65/109) of isolates. A total of 10.1% (11/109) of KPC-positive isolates were simultaneously producing both CTX-M-14-type ESBL and DHA-1-type plasmid-mediated AmpC enzymes. GIM-1-type or VIM-1-type MBL, OXA-type carbapenemases were detected in 9.2% (10/109) and 10.1% (11/109) of isolates, respectively. Table 1. Microbiological activities of various antimicrobial agents against 109 carbapenem-resistant K. pneumoniae isolates In this study, we have reported for the first time the frequency of KPC-2-type carbapenemases with or without CTX-M-type ESBLs among carbapenem-resistant K. pneumoniae isolates in China. In our hospital, the incidence of carbapenem-resistant K. pneumoniae isolates showed a significant increase from 0.9% in 2005 to 12.9% in 2009. These results suggested that the rapidly increased prevalence of carbapenem resistance among K. pneumoniae isolates in our hospital could be the consequence of the failure to control the spread of these strains. Therefore, prompt detection of carbapenemase-producing Enterobacteriaceae isolates, active antibiotic resistance surveillance, and strict implementation of infection control measures are critical to avoid the rapid spread or outbreaks by these multidrug-resistant or pan-drug-resistant isolates in health care-associated facilities (3, 5–7, 9).

Comparison of Adhesin Genes and Antimicrobial Susceptibilities between Uropathogenic and Intestinal Commensal Escherichia coli Strains

The presence of adhesins is arguably an important determinant of pathogenicity for Uropathogenic Escherichia coli (UPEC). Antimicrobial susceptibilities were tested by agar dilution method, fifteen adhesin genes were detected by polymerase chain reaction, and multilocus sequence typing (MLST) was analyzed in 70 UPEC isolates and 41 commensal E. coli strains. Extended-spectrum β-lactamase (ESBL) was determined with confirmatory test. The prevalence of ESBL-producers in UPEC (53%, 37/70) was higher than the commensal intestinal isolates (7%, 3/41), and 97% (36/37) of the ESBL-producing UPEC harbored bla CTX-M genes. afa was present in 36% (10/28) UPEC isolates from recurrent lower urinary tract infection (UTI), and none in the acute pyelonephritis, acute uncomplicated cystitis or commensal strains (P<0.0001). papG was detected in 28% (20/70) of UPEC isolates, while 5% (2/41) of the commensal strains were papG positive (P = 0.0025), and the prevalence of papG was significantly higher in acute pyelonephritis group (71%) than the other two UTI groups (P<0.0001). The prevalence of flu, yqi, yadN and ygiL was significantly higher in UPEC isolates than in the commensal strains. ESBL-producing UPEC showed a lower prevalence of adhesin genes compared with non-ESBL-producing strains. The MLST profiles were different between UPEC and commensal strains, with ST131 (19%, 13/70) and ST10 (20%, 8/41) being the most common MLSTs, respectively. This study demonstrated that several adhesin genes were more prevalent in UPEC isolates than in commensal E. coli, and afa may be associated with recurrent lower UTI whereas papG is more frequently associated with acute pyelonephritis.

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

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 carbapenem-resistant 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 µg ml(-1) and 4 µg 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.

Occurrence of False Positive Results for the Detection of Carbapenemases in Carbapenemase-Negative Escherichia coli and Klebsiella pneumoniae Isolates

Adequate detection of the production of carbapenemase in Enterobacteriaceae isolates is crucial for infection control measures and the appropriate choice of antimicrobial therapy. In this study, we investigated the frequency of false positive results for the detection of carbapenemases in carbapenemase-negative Escherichia coli and Klebsiella pneumoniae clinical isolates by the modified Hodge test (MHT). Three hundred and one E. coli and K. pneumoniae clinical isolates were investigated. All produced extended spectrum β-lactamases (ESBLs) but were susceptible to carbapenems. Antimicrobial susceptibility testing was performed by the disk diffusion and agar dilution methods. The MHT was performed using the standard inoculum of test organisms recommended by the CLSI. Genes that encoded ESBLs and carbapenemases were identified by PCR and DNA sequencing. Among the 301 clinical isolates, none of the isolates conformed to the criteria for carbapenemase screening recommended by the CLSI. The susceptibility rates for imipenem, meropenem, and ertapenem all were 100.0%, 100.0%, and 100.0%, respectively. Of the 301 E. coli and K. pneumoniae isolates, none produced carbapenemase. The MHT gave a positive result for 3.3% (10/301) of the isolates. False positive results can occur when the MHT is used to detect carbapenemase in ESBL-producing isolates and clinical laboratories must be aware of this fact.

Mechanisms of Tigecycline Resistance among Klebsiella pneumoniae Clinical Isolates

ABSTRACT Of 26 tigecycline-nonsusceptible Klebsiella pneumoniae (TNSKP) clinical isolates, 25 had nonsynonymous mutations in ramR and/or acrR (23 in ramR and 10 in acrR). Eight TNSKP isolates possessed overexpression of ramA, acrB, rarA, and oqxB simultaneously, while 8 and 1 TNSKP strains had upregulation of ramA and acrB and of rarA and oqxB, respectively. Thus, resistance mechanisms of 9 TNSKP isolates cannot be explained by the present pathways. This study underscores the role of RamA in TNSKP and suggests the presence of novel tigecycline resistance mechanisms.