CTX-M Type ESBL Research Articles

Novel Extended-spectrum β-Lactamase inShigella sonnei

To the Editor: A 38-year-old French man with a history of chronic juvenile arthritis was referred to the Necker-Enfants Malades University hospital (Paris, France) with a dysenteric syndrome. The patient had returned the day before from a 1-month stay in Port-au-Prince, Haiti, where he spent most of his time in close contact with young children from an orphanage, most of whom had diarrhea. Clinical examination at admission showed fever (39°C), chills, diffuse abdominal pain, bloody diarrhea, and vomiting. The patient received ceftriaxone, which was stopped on day 4 because initial blood and stool cultures were negative for pathogens and clinical signs had completely resolved. Ten days later, he reported the recurrence of diarrhea without fever. A novel stool culture grew Shigella sonnei. An extended-spectrum β-lactamase (ESBL) was detected by double-disk synergy test; the isolate was also resistant to aminoglycosides (except amikacin), tetracycline, and cotrimoxazole. The strain was susceptible to fluoroquinolones and fosfomycin. It also appeared susceptible to azithromycin (MIC 4 μg/mL), although azithromycin MIC for Shigella spp. should be interpreted with caution (1). The patient was successfully treated with azithromycin at a dose of 500 mg/d for 5 days. Azithromycin was preferred to fluoroquinolones to avoid the risk for tendinopathy because of the patient’s history of chronic juvenile arthritis and because this antimicrobial agent was shown to be effective in the treatment of shigellosis caused by multidrug-resistant strains (2). To identify the molecular basis of this ESBL, a series of PCR primers were used for detection of TEM-, SHV-, or CTX-M–type ESBL (3). Only the TEM PCR showed positive results. Sequencing of 2 independent PCR products showed a new allele (www.lahey.org/studies/temtable.asp). Analysis of the deduced amino acid sequence allowed characterization of TEM-137, derived from TEM-1 with 2 substitutions, Arg-16→Ser and Glu-240→Arg. This ESBL (and resistance to aminoglycosides and tetracyclines) was easily transferred to Escherichia coli J53-2 by conjugation. MICs of β-lactams alone or in association with clavulanic acid, were determined by E-test, according to manufacturer’s instructions (AB Biodisk, Solna, Sweden). High-level resistance to ceftazidime (MIC 32 μg/mL) and intermediate resistance to cefotaxime (MIC 8 μg/mL) were observed; the strain remained susceptible to cefepime and imipenem (MIC 0.5 and 0.25 μg/mL, respectively). Clavulanic acid did not restore susceptibility to ceftazidime (MIC 4 μg/mL) but did restore susceptibility to cefotaxime (MIC 0.5 μg/mL). With clavulanic acid, the MIC of cefepime was 0.06 μg/mL. ESBL in S. sonnei is rare worldwide. In Argentina, a CTX-M-2 was found in an isolate of S. sonnei resistant to cefotaxime but not to ceftazidime (4). In South Korea, TEM-15, TEM-17, TEM-19, TEM-20, TEM-52, and CTX-M-14 were characterized in S. sonnei (5); TEM-52 and CTX-M-14 were also widely distributed, particularly in Salmonella spp. (6,7). In Turkey, an isolate of S. sonnei producing CTX-M-3 was reported (8). In Hong Kong, sequencing of 2 S. sonnei isolates showed the presence of CTX-M-14 and CTX-M-15 (9). Finally, in Bangladesh, 2 isolates of S. sonnei with a class A ESBL were reported; they were not characterized at the molecular level, but the resistance phenotypes suggested a CTX-M type (10). In our case, little information on antimicrobial drug resistance could be obtained from Haiti because no systematic investigation on resistance in Enterobacteriaceae is performed. Nevertheless, the emergence of TEM-137 (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AM286274,term_id:110350793,term_text:AM286274}}AM286274) harbored by this imported S. sonnei isolate clearly demonstrates that ESBL-associated shigellosis has emerged in Haiti and that potentially large and severe shigellosis outbreaks could occur, for which the use of azithromycin could be beneficial, as illustrated in our patient. Because treating shigellosis is becoming problematic, it is essential to focus on prevention measures such as simple rules of personal hygiene that might drastically decrease the risk of transmission.

CTX-M Extended-spectrum β-Lactamases, Washington State

To the Editor: The CTX-M–type β-lactamases are non-TEM and non-SHV plasmid-mediated, class A, extended-spectrum β-lactamases (ESBLs). The CTX-M–type β-lactamases have recently emerged as the most common type of ESBLs, with a global distribution (1). In contrast, the CTX-M–type ESBLs are rarely reported in the United States and have not been identified in pathogens isolated from infected patients with gastroenteritis. We screened 637 Salmonella and 126 Shigella isolates, collected in the state of Washington during 2003–2004, for CTX-M–type β-lactamases. Of these, 60 Salmonella isolates that exhibited an ESBL phenotype were further characterized by PCR for TEV, SHV, CTM-X, and CMY. All were positive for the CMY-2 or TEM-1 β-lactam genes. One Shigella sonnei isolate (WA7593), cultured from a fecal specimen in August 2004, tested positive with an ESBL confirmatory disk diffusion panel (ceftazidime 24 mm, ceftazidime/clavulanate 32 mm, cefotaxime 14 mm, and cefotaxime/clavulanate 34 mm; [2]). The patient had recently traveled to Pakistan and likely became ill there and returned to the United States while still sick. The transfer of extended-spectrum cephalosporin resistance was tested by conjugation to Escherichia coli J53 aziR (3). The MIC for S. sonnei WA7593 and its transconjugant, WA7593TC1, were tested by using the E-test (AB Biodisk, Solna, Sweden). Both strains were resistant to cefotaxime and susceptible to ceftazidime and showed almost the same antimicrobial susceptibility patterns as β-lactam antimicrobial drugs (Table). Table MICs of antimicrobial drugs for Shigella sonnei clinical isolate WA7593 and its transconjugant WA7593TC1 The type of ESBL produced by these strains was determined by using PCR specific for TEM and CTX-M (4,5). Both strains were PCR positive for TEM and CTX-M. The TEM type PCR products were then sequenced and identified as TEM-1; no variation was found on the promoter region of blaTEM-1. The entire sequence of bla CTX-M from WA7593 was then sequenced (1), and the product showed 100% homology with blaCTX-M-15 (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AY960984,term_id:63029819,term_text:AY960984}}AY960984). The mobile element associated with the transfer of blaCTX-M-15 was investigated by sequencing the flanking regions. PCRs were performed with primers from the internal regions of bla CTX-M gene and primers for insertion sequences ISEcp1 and IS903 (4,5). Positive PCR products were obtained with primers ISEcp1F and CTX2 (943 bp); no amplified product was produced with primers CTX1 and IS903R. Sequencing of a 943-bp amplicon showed that blaCTX-M15 was flanked upstream by an ISEcp1-like element. The presence of an integron in S. sonnei WA7593 and WA7593TC1 was investigated by using integron-specific primers hep35 and hep36 (2). Only S. sonnei WA7593 produced a PCR product. This finding suggests that the transmission of blaCTX-M15 is not by integron-mediated transfer. A further 162 Shigella spp. and 260 Salmonella spp. isolated from 2003 through 2005 were also screened for ESBL production; no further isolates were identified. The presence of a CTX-M–type, ESBL-producing isolate is rarely reported in the United States. The only other reference was from a multistate study in 2001–2002 that identified CTX-M type from E. coli isolates from urine, sputum, and blood (6). No further reports about CTX-M–producing organisms have been disseminated. Our investigation suggests that CTX-M–type ESBLs may spread throughout the United States through infected travelers. This finding is notable because S. sonnei is a common enteric pathogen. Our results further emphasize that travelers from others parts of the world can introduce highly mobile and clinically important resistance mechanisms into the community. The spread of CTX-M ESBLs may be faster and more widespread than previously thought; therefore, CTX-M type should be taken seriously as a surveillance target in the United States, especially in patients with a history of travel outside North America.

Extended-spectrum β-Lactamase–producingEnterobacteriaceae, Central African Republic

Extended-spectrum β-Lactamase–producing<i>Enterobacteriaceae</i>, Central African Republic

Resistance of strains producing extended-spectrum β-lactamases and genotype distribution in China

To investigate the resistance of Escherichia coli and Klebsiella pneumoniae producing extended-spectrum beta-lactamases (ESBLs) and the genotyping of ESBLs in China. MICs of 12 antibiotics against 50 strains (by random selection) of ESBLs-producing E. coli and K. pneumoniae were determined by E-test. The genotypes of ESBLs were analyzed by PCR, DNA sequencing and isoelectric focusing. The susceptibility rate of 50 isolates was 100% in imipenem, 60%-80% in cefoperazone/sulbactam, ceftazidime and piperacillin/tazobactam, and lower in other antimicrobial agents tested. Only 6.0% of the isolates were sensitive to cefotaxime. Four hundred and forty-seven of 509 isolates had been confirmed the genotype of ESBLs. Four hundred and sixteen strains produced only one type of ESBLs, including CTX-M-14 (271 strains), CTX-M-3 (70 strains), CTX-M-24 (35 strains), CTX-M-22 (8 strains), CTX-M-15 (4 strains), CTX-M-9 (4 strains), CTX-M-28 (3 strains), CTX-M-12 (1 strain), CTX-M-13 (1 strain), CTX-M-27 (1 strain), CTX-M-29 (1 strain), SHV-12 (10 strains), SHV-5 (4 strains), SHV-2 (2 strains), and SHV-9 (1 strain). Thirty isolates carried two or three types of ESBLs, and producing CTX-M-14 and CTX-M-3 together were the most common type. The resistance of E. coli and K. pneumonia producing ESBLs in China was a serious issue and CTX-M type ESBLs were the most common genotype. CTX-M-14 was the predominant genotype. Some isolates produced two or three ESBLs.

Prevalence and molecular epidemiology of CTX-M extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae in Russian hospitals.

A total of 904 consecutive nosocomial isolates of Escherichia coli and Klebsiella pneumoniae collected from 28 Russian hospitals were screened for production of extended-spectrum beta-lactamases (ESBLs). The ESBL phenotype was detected in 78 (15.8%) E. coli and 248 (60.8%) K. pneumoniae isolates. One hundred fifteen isolates carried the genes for CTX-M-type beta-lactamases, which, as shown by PCR-restriction fragment length polymorphism analysis, were distributed into the two genetic groups of CTX-M-1 (93%)- and CTX-M-2 (7%)-related enzymes. Isolates producing the enzymes of the first group were found in 20 hospitals from geographically distant regions of the country and were characterized by considerable diversity of genetic types, as was demonstrated by enterobacterial repetitive consensus PCR typing. Within this group the CTX-M-3 and the CTX-M-15 beta-lactamases were identified. In contrast, the enzymes of the CTX-M-2 group (namely, CTX-M-5) were detected only in eight clonally related E. coli isolates from a single hospital. Notably, the levels of resistance to ceftazidime were remarkably variable among the CTX-M producers. This study provides further evidence of the global dissemination of CTX-M type ESBLs and emphasizes the need for their epidemiological monitoring.

EHV001 Molecular characterisation of a 110 kb LEE containing PAI in the emerging enterohaemorrhagic E. coli (EHEC) strains of serotype O103:H2

The aim of this study was to reveal phenotype/genotype characteristics of verotoxigenic Escherichia coli (VTEC) and multidrug resistant E. coli in food products of animal origin confiscated as illegal import at Austrian, German and Slovenian airports. VTEC isolates were obtained by using ISO guidelines 16654:2001 for O157 VTEC or ISO/ TS13136:2012 for non-O157 VTEC, with additional use of the RIDASCREEN® Verotoxin immunoassay. The testing of 1526 samples resulted in 15 VTEC isolates (1.0%) primarily isolated from hard cheese from Turkey and Balkan countries. Genotyping for virulence by using a miniaturized microarray identified a wide range of virulence determinants. One VTEC isolate (O26:H46) possessing intimin (eae) and all other essential genes of Locus of Enterocyte Effacement (LEE) was designated as enterohemorrhagic E. coli (EHEC). None of the other VTEC strains belonged to serogroups O157, O145, O111, O104 or O103. VTEC strains harbored either stx1 (variants stx1a or stx1c) or stx2 (variants stx2a, stx2b, stx2a/d or stx2c/d) genes. Pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) demonstrated high genetic diversity and identified three new sequence types (STs): 4505, 4506 and 4507. Food samples collected from the Vienna airport were also tested for E. coli quantities using the ISO 16649:2001, and for detection of multidrug resistant phenotypes and genotypes. The resulting 113 commensal E. coli isolates were first tested in a pre-screening against 6 selected antimicrobials to demonstrate multidrug resistance. The resulting 14 multidrug resistant (MDR) E. coli isolates, representing 0.9% of the samples, were subjected to further resistance phenotyping and to microarray analyses targeting genetic markers of antimicrobial resistance and virulence. Genotyping revealed various combinations of resistance determinants as well as the presence of class 1, class 2 integrons. The isolates harbored 6 to 11 antibiotic resistance genes as well as 1 to 14 virulence genes. In this panel of 14 MDR E. coli two strains proved to carry CTX-M type ESBLs, and one single isolate was identified as enteropathogenic E. coli (EPEC). In general, isolates carrying a high number of resistance determinants had lower number of virulence genes and vice versa. In conclusion, this first pilot study on the prevalence of VTEC and of MDR/ESBL E. coli in illegally imported food products of animal origin suggests that these strains could represent reservoirs for dissemination of potentially new types of pathogenic and MDR E. coli in Europe.