Plasmid-mediated Extended-spectrum Β-lactamases Research Articles

Performance of Ceftazidime-Avibactam 30/20-μg and 10/4-μg Disks for Susceptibility Testing of Enterobacterales and Pseudomonas aeruginosa.

Ceftazidime-avibactam, a new β-lactam-β-lactamase inhibitor combination, is active against multidrug-resistant Enterobacterales and Pseudomonas aeruginosa isolates and has became available for clinical use in China in the latter half of 2019. In this study, we evaluated the performance of the disk diffusion test with ceftazidime-avibactam 10/4-μg and 30/20-μg disks, compared with the reference broth microdilution method, with a collection of 467 Enterobacterales and 182 P. aeruginosa nonduplicate clinical isolates. The results of antimicrobial susceptibility testing indicated that the categorical agreement (CA) of ceftazidime-avibactam 10/4-μg disk testing for all tested Enterobacterales isolates was 99.8%, with 0.5% very major errors (VMEs) and no major error (ME). The CA of ceftazidime-avibactam 10/4-μg disk testing for all tested P. aeruginosa isolates was 87.9%, with 15.5% MEs and no VME. The CA of ceftazidime-avibactam 30/20-μg disk testing for all tested Enterobacterales isolates was 99.4%, with 1.5% VMEs and no ME. The CA of ceftazidime-avibactam 30/20-μg disk testing for all tested P. aeruginosa isolates was 91.8%, with 2.5% VMEs and 9.9% MEs. Overall, ceftazidime-avibactam 10/4-μg disk testing showed superior performance and was more suitable for assessment of the susceptibility of Enterobacterales and P. aeruginosa isolates. IMPORTANCE Multidrug-resistant Enterobacterales and P. aeruginosa strains have become a global public threat, with the emergence and prevalence of plasmid-mediated extended-spectrum β-lactamases (ESBLs), AmpC cephalosporinases, and carbapenemases disseminated worldwide. Ceftazidime-avibactam, which is commercially available, has shown excellent in vitro activity against multidrug-resistant and carbapenem-resistant Enterobacterales and P. aeruginosa isolates. Moreover, ceftazidime-avibactam has shown promise in treating infections caused by multidrug-resistant and carbapenem-resistant isolates. The disk diffusion test for ceftazidime-avibactam is the most common antimicrobial susceptibility testing method in most laboratories in China. The accurate detection of ceftazidime-avibactam susceptibility is of great significance for the rational clinical application of drugs. Here, we evaluated the performance of the ceftazidime-avibactam 10/4-μg and 30/20-μg disk diffusion tests, compared with the reference broth microdilution method, with clinical Enterobacterales and P. aeruginosa isolates.

Effect of challenge dose of plasmid-mediated extended-spectrum β-lactamase and AmpC β-lactamase producing Escherichia coli on time-until-colonization and level of excretion in young broilers

Plasmid-mediated extended-spectrum β-lactamase and AmpC β-lactamase (ESBL/pAmpC) producing bacteria are present at all levels of the broiler production pyramid. Young birds can be found positive for ESBL/pAmpC-producing Escherichia coli shortly after arrival at farm. The aim of this study was to determine the effect of different challenge doses of ESBL/pAmpC-producing E. coli on time-until-colonization and the level of excretion in young broilers. One-day-old broilers (specific-pathogen free (SPF) and conventional Ross 308) were housed in isolators and challenged with 0.5 ml ESBL/pAmpC-producing E. coli strains of varying doses (101–105 CFU/ml). Presence and concentration (CFU/gram feces) of ESBL/pAmpC-producing E. coli and total E. coli were determined longitudinally from cloacal swabs, and in cecal content 72 h after challenge. Higher challenge doses resulted in shorter time-until-colonization. However, even the lowest dose (101 CFU/ml) resulted in colonization of the broilers which excreted >106 CFU/gram feces 72 h after inoculation. Conventional broilers were colonized later than SPF broilers, although within 72 h after challenge all broilers were excreting ESBL/pAmpC-producing E. coli. A probabilistic model was used to estimate the probability of colonization by initial inoculation or transmission. The higher the dose the higher the probability of excreting ESBL/pAmpC-producing E. coli as a result of inoculation. In conclusion, low initial doses of ESBL/pAmpC-producing E. coli can result in rapid colonization of a flock. Interventions should thus be aimed to eliminate ESBL/pAmpC-producing bacteria in the environment of the hatchlings and measures focusing at reducing colonization and transmission of ESBL/pAmpC-producing E. coli should be applied shortly after hatching.

Emergence of Enterobacteriaceae co-producing CTX-M-15, ArmA and PMQR in Poland.

Plasmid-mediated extended-spectrum β-lactamases (ESBLs), 16S rRNA methylases and quinolone resistance mechanisms (PMQRs) are well-known agents conferring resistance to more than 1 antimicrobial in its group. The accumulation of these agents poses, therefore, a serious risk to public health. The objective of this study was to investigate the presence of common ß-lactamases and 16S rRNA methylases in Qnr-producing Enterobacteriaceae and their genetic relatedness. We examined 18 Qnr-producing isolates (Klebsiella pneumoniae n = 8, Enterobacter cloacae n = 6 and Escherichia coli n = 4) selected from a collection of 215 ciprofloxacin-resistant strains obtained from patients in a 1030-bed tertiary hospital from 1 March to 31 August 2010. The antibiotics minimum inhibitory concentration (MIC) was determined by E-test. The detection of common ß-lactamases, 16S rRNA methyltransferases and PMQR genes was performed by polymerase chain reaction (PCR) and sequencing. Genetic relatedness was assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). All the isolates tested were susceptible to carbapenems and colistin, while 16 were multidrug-resistant. Thirteen, 2 and 2 isolates carried qnrB1, qnrA1 and qnrS1, respectively. Ten of 13 qnrB1-positive Enterobacteriaceae also carried genes encoding for aac(6')-Ib-cr and at least 1 ESBL. The blaCTX-M-15 gene was the most common ESBL. The most prevalent combination of genes was qnrB1+aac(6')-Ib-cr+blaTEM-1+blaCTX-M-15. Two isolates of K. pneumoniae and E. cloacae were found to bear multiple extended range resistance traits: ArmA, CTX-M-15, QnrB1, and AAC (6')-Ib-cr. The PFGE showed that most of the isolates exhibited individual DNA patterns, whilst MLST assigned K. pneumoniae (n = 8) to 5 sequence types (STs) (ST15, ST323, ST336, ST147, and ST525), E. coli (n = 4) to 2 (ST131 and ST1431) and E. cloacae (n = 5) to 4 (ST90, ST89, ST133, and the novel ST407). Our findings reveal the accumulation of resistance traits and their important role in spreading of multiresistant bacteria among hospitalized patients.

Detection of Plasmid-Mediated β-Lactamase Genes and Emergence of a Novel AmpC (CMH-1) in Enterobacter cloacae at a Medical Center in Southern Taiwan.

The plasmid-mediated extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases in Enterobacter spp. have increasingly been reported. In this study, we investigated the prevalence of the plasmid-mediated β-lactamases in Enterobacter cloacae from bloodstream isolates at a medical center in southern Taiwan. ESBL and ampC genes were detected by PCRs and DNA sequencing. Conjugation experiments were conducted to confirm the transferability of the genetic resistance trait. Among 41 non-repetitive blood isolates of cefuroxime-resistant E. cloacae, eight isolates exhibited ESBL phenotype confirmed by double-disk synergistic tests. Nearly all the strains were susceptible to carbapenems. The prevalence rate of the plasmid-mediated blaampC genes was 73% (30/41), including one blaDHA-1, one blaMIR-6, two novel blaCMH-1 genes and other blaACT-like genes. Coexistence of plasmid-mediated blaACT and ESBL genes (10 with blaSHV-12 and one with blaCTX-M-3) was observed. Successful transmissions of the blaACT and blaCMH-1 were demonstrated in some transconjugants. The inducible or derepressed CMH-1 had expanded activity of isolates versus ceftazidime. Enterobacterial repetitive intergenic consensus (ERIC)-PCR analysis and pulsotype showed distinct patterns suggesting non-clonal relationship. In conclusion, plasmid-mediated blaACT-like ampC genes in E. cloacae isolates have been highly prevalent in southern Taiwan and may continue genetic evolution, contributing to the complexities in antibiotic-resistant mechanisms.

Widespread distribution of CTX-M and plasmid-mediated AmpC β-lactamases in Escherichia coli from Brazilian chicken meat.

The dissemination of plasmid-mediated antimicrobial resistance genes may pose a substantial public health risk. In the present work, the occurrences of blaCTX-M and plasmid-mediated ampC and qnr genes were investigated in Escherichia coli from 16 chicken carcasses produced by four commercial brands in Brazil. Of the brands tested, three were exporters, including one of organic chicken. Our study assessed 136 E. coli isolates that were grouped into 77 distinct biotypes defined by their origin, resistance profiling, the presence of β-lactamase and plasmid-mediated quinolone resistance genes and enterobacterial repetitive intergenic consensus-polimerase chain reaction typing. The blaCTX-M-15, blaCTX-M-2 and blaCTX-M-8 genes were detected in one, 17 and eight different biotypes, respectively (45 isolates). Twenty-one biotypes (46 isolates) harboured blaCMY-2. Additionally, blaCMY-2 was identified in isolates that also carried either blaCTX-M-2 or blaCTX-M-8. The qnrB and/or qnrS genes occurred in isolates carrying each of the four types of β-lactamase determinants detected and also in oxyimino-cephalosporin-susceptible strains. Plasmid-mediated extended-spectrum β-lactamase (ESBL) and AmpC determinants were identified in carcasses from the four brands tested. Notably, this is the first description of blaCTX-M-15 genes in meat or food-producing animals from South America. The blaCTX-M-8, blaCTX-M-15 and blaCMY-2 genes were transferable in conjugation experiments. The findings of the present study indicate that plasmid-mediated ESBL and AmpC-encoding genes are widely distributed in Brazilian chicken meat.

Genetic and structural insights into plasmid-mediated extended-spectrum β-lactamase activity of CTX-M and SHV variants among pathogenic Enterobacteriaceae infecting Indian patients

Resistance to third-generation cephalosporins (3GCs) mediated by extended-spectrum β-lactamases (ESBLs) in pathogenic Enterobacteriaceae is considered a major public health threat in India. This study deals with the detection of plasmid-mediated blaCTX-M, blaSHV and blaOXA genes, understanding their contribution to the ESBL phenotype, and their molecular interaction with 3GCs. More than 87% of isolates showed 3GC resistance, with ESBL production in 60.0% of Escherichia coli and 47.7% of Klebsiella pneumoniae. Molecular characterisation revealed the presence of blaCTX-M-15 (29.8%), blaCTX-M-truncated (1.3%), blaCTX-M-27 (0.7%), blaSHV-1 (20.5%), blaSHV-11 (2.0%), blaSHV-42 (0.7%) and blaOXA-1 (9.9%), among which blaCTX-M variants and blaSHV-42 were ESBLs. Phylogenetic analysis predicted strong selection pressure on all blaCTX-M variants, blaSHV-11 and blaSHV-42. The instability index and Gibbs free folding energy change (ΔΔG) predicted decreased stability of SHV-11 and SHV-42. Mutations of CTX-M-truncated, SHV-11 and SHV-42 located in the core region of the enzymes were found to be functional/pathogenic in nature. The catalytic pockets of CTX-M-15 and SHV-42 had the greatest molecular surface area, which might explain their expanded substrate spectrum towards oxyimino-cephalosporins. Molecular dynamics analysis indicated different structural flexibility of CTX-M-truncated compared with the other enzymes. Amino acid alterations resulted in a change of orientation of catalytic residues of class A β-lactamases that might affect their catalytic processes. Molecular interactions revealed higher catalytic efficiency (ΔG and Km) of CTX-M-15, CTX-M-truncated, CTX-M-27, SHV-11 and SHV-42 compared with their respective wild-types. This study provides useful insights into ESBL production of pathogenic Enterobacteriaceae in India that might help in the development of new antibiotics.

In Memoriam: John P. Quinn, MD

In Memoriam: John P. Quinn, MD

Co-production of SFO-1 and DHA-1 -lactamases and 16S rRNA methylase ArmA in clinical isolates of Klebsiella pneumoniae

To investigate the epidemiology and genetic characteristics of the plasmid-mediated extended-spectrum β-lactamase (ESBL) gene bla(SFO-1) in clinical isolates of ESBL-producing Klebsiella pneumoniae and Escherichia coli. The prevalence of the bla(SFO-1) gene was examined by PCR amplification. Conjugation and transformation experiments were performed and the presence of antimicrobial resistance determinants was investigated by the PCR method. The genetic environments of the bla(SFO-1) and armA genes were determined by direct sequencing of plasmid pHS20. Plasmids were typed by PCR-based replicon typing. PFGE and multilocus sequence typing were performed on SFO-producing strains. Of 158 ESBL-producing strains of K. pneumoniae, 3 (1.9%) carried the bla(SFO-1) gene. All of the SFO-producing isolates belonged to the ST11 epidemic clone, with a single PFGE type and had high-level resistance to third-generation cephalosporins, aminoglycosides and ciprofloxacin. The bla(SFO-1) gene was co-transferred with the armA, aac(6')-Ib-cr and bla(TEM-1) genes by transformation, whereas the armA, bla(DHA-1) and qnrB4 genes were co-transferred by conjugation. The armA genes were located within the composite transposon Tn1548 on two different plasmids in the strain 08-129. The bla(SFO-1) gene was located upstream of an ampR gene from the coding region and flanked by two inverted repeats of IS26. Plasmids carrying bla(DHA-1) were identified as IncFII, while the bla(SFO-1)-bearing plasmids were non-typeable. Although SFO-1 is a low-occurrence ESBL, it has been captured by a plasmid accumulating multiple resistance determinants including armA and aac(6')-Ib-cr, and accompanied by a large DHA-1-bearing IncFII plasmid in a prevalent K. pneumoniae ST11 clone.

Study of Plasmid-Mediated Extended-Spectrum β-Lactamase-Producing Strains of Enterobacteriaceae, Isolated from Diabetic Foot Infections in a North Indian Tertiary-Care Hospital

This study evaluated the incidence and factors responsible for plasmid-mediated extended-spectrum β-lactamase (ESBL) infection among patients with diabetic foot ulcer (DFU). A prospective study was conducted on 162 DFU inpatients treated in a multidisciplinary-based diabetes and endocrinology center at Jawaharlal Nehru Medical College of Aligarh Muslim University, Aligarh, India, during the period of December 2008-March 2011. Detailed history and patient's profile, grade of DFU, co-morbidities and complications, laboratory data, and final outcome were collected. Standard methods were used for culture identification, sensitivity testing, and ESBL detection. Polymerase chain reaction for bla genes was performed, and the risk factors for bla gene positivity were determined by univariate analysis with 95% confidence interval. In total, 127 (78.3%) Enterobacteriaceae members were isolated. The most common isolate was Escherichia coli (71; 55.9%), followed by Klebsiella sp. (33; 25.9%) and Proteus sp. (13; 10.2%). By phenotypic methods, 67.8% were ESBL producers. In the molecular detection of ESBLs, 81.9% were found to be positive for the bla gene, of which bla(CTX-M) showed 81.8% positivity, followed by bla(TEM) (50%) and bla(SHV) (46.9%). In a univariate analysis, bla gene-positive status was associated with low-density lipoprotein-cholesterol (>100 mg/dL) (P<0.004, odds ratio 13.4, relative risk 8.65) and triglycerides (>200 mg/dL) (P<0.003, odds ratio 6.5, relative risk 4.11). ESBL constitutes a major threat to currently available β-lactam therapy, leading to complications in DFUs. Aminoglycosides, cephalosporin, and β-lactam inhibitor drugs would probably be more appropriate empirical agents after establishing the patient's history of previous antibiotic use. The detection of ESBL should be done on a routine basis.

When Carbapenem-Hydrolyzing β-Lactamase KPC Meets Escherichia coli ST131 in France

Carbapenem resistance in Escherichia coli isolates, though rare, can be attributed to outer membrane protein deficiency coupled with plasmid-mediated extended-spectrum β-lactamases (ESBLs) or class C β-lactamases ([2][1], [10][2], [11][3]) or to carbapenemases. The latter can be either metallo-β-

Prevalence and molecular characterization of extended-spectrum β-lactamase-producing Klebsiella pneumoniae in Riyadh, Saudi Arabia

BACKGROUND AND OBJECTIVES:Reports on extended-spectrum β-lactamases (ESBL) production by Enterobacteriaceae, and especially in Klebsiella pneumoniae, are few in Saudi Arabia. Therefore, we determined the prevalence of ESBL in K pneumoniae from Riyadh and characterized the predominant β-lactamase gene in these isolates.METHODS:A total of 400 K pneumoniae samples were isolated from two hospitals in Riyadh during 2007 and screened for production of ESBL using ESBL-E-strips and combined disk methods. PCR assay was used to detect blaTEM, blaSHV, and blaCTX-M genes.RESULTS:Phenotypic characterization identified a high ESBL rate of 55% of K pneumoniae isolates. ESBL-producing K pneumoniae were PCR positive for SHV, TEM and CTX-M β-lactamase genes with prevalences 97.3%, 84.1% and 34.1%, respectively. Within the CTX-M family, two groups of enzymes, CTX-M-1 and CTX-M-9-like genes were found with prevalences of 60% and 40%, respectively.CONCLUSIONS:This study confirms the high rate of ESBL in K pneumoniae clinical isolates in hospitals in Riyadh. This study demonstrates the worldwide spread of blaCTX-M genes. This first report of the presence of the blaCTX-M gene in clincial isolates in Saudi Arabia is evidence of the continuing worldwide spread of this gene.

Study on plasmid-mediated extended spectrum β-lactamases and their resistance phenotypes in Shigella

Objective To discuss the characteristics of extended-spectrum beta-lactamases(ESBLs)-producing Shigella and the relation between them and drug-resistance plasmid. Methods The suspicious ESBLs-producing isolates were screened by K-B disc diffusion method, and the ESBLs-producing strains were confirmed by confirmatory test recommended by the National Committee for Clinical Laboratory Standards. Furthermore, the partial blageneof these isolates were detected by PCR using universal primers for TEM, SHV, CTX-M-1 group, CTX-M-2 group and CTX-M-9 group, respectively. The entire blaCTX-M-9 and blaTEM were amplified by PCR using the primers outside the open reading frame (ORF) of these β-1actamases and products were directly sequenced. The conjugation experiment was performed to determine whether the resistance was transferable. Minimal inhibitory concentration (MIC) was detected with double agar dilution method. Results Of the 275 isolates, 12 strains were identified as ESBLs producers. Among them, 8 strains were CTX-M-14 carriers and 4 strains were CTX-M-3 carriers. All ESBLs-producing isolates are positive for plasmid conjugative transfer test. The transconjugants are only resistance to betalactams. Conclusions High resistance to beta-laetams in Shigella is caused by production of ESBLs in the local area. The ESBLs-produeing isolates can transfer the drug resistance through lateral transfer of plasmid. Key words: Shigella; beta-Lactamases; Plasmids; Drug resistance, bacterial

CTX-M β-lactamase–producingEscherichia coliin Long-term Care Facilities, France

To the Editor: In long-term care facilities, most endemic infections affect respiratory and urinary tracts, as well as skin and soft tissues (1–3). Infection and colonization by antimicrobial-resistant organisms, in particular those producing plasmid-mediated extended-spectrum β-lactamases (ESBL), are common in long-term care facilities (4). Since 1984, ESBL-producing Enterobacteriaceae have spread among French hospitals; within Parisian public hospitals (Assistance Publique, Hopitaux de Paris), ESBL-producing Escherichia coli is the most frequent species found, representing 49.5% of 220 Enterobacteriaceae isolated in 2002, mostly in urinary tract infections (5). Among ESBL-producing Enterobacteriaceae, CTX-M–type β-lactamases confer a higher level of resistance to cefotaxime and ceftriaxone than to ceftazidime. CTX-M–producing strains are endemic in Latin America, Japan, and certain parts of Eastern Europe; in contrast, these strains are emerging in France, Western Europe, and the United States (6). We report the first documented outbreak of CTX-M–producing E. coli infection in a long-term care facility in France. Our hospital is an 800-bed institution with 300 beds for long-term patients distributed among three units located in two buildings. The outbreak occurred in a 35-bed unit and involved 26 of 47 hospitalized patients from October 2001 to March 2003. This facility hosts patients for extended periods of time or permanently. The index case was identified in October 2001; the patient had a urinary tract infection attributable to an ESBL-producing E. coli, which showed resistance patterns not previously found in our hospital. Three new cases were detected within the following 2 months, and all patients had urinary tract infection with the same pattern of resistance. In January 2002, patients were screened for ESBL-producing strains by rectal swabbing and urine culture. The results showed E. coli with a high level of resistance to amoxicillin and ticarcillin (MIC > 128 µg/mL), partial restoration of susceptibility to these agents by addition of clavulanic acid (MIC = 16–32 µg/mL), and higher resistance to cefotaxime (MIC > 128 µg/mL) than to ceftazidime (MIC = 32–64 µg/mL.) A cephalosporin/co-amoxiclav synergy test was positive, which suggests a CTX-M ESBL. Strains were also resistant to ciprofloxacin (MIC 64 µg/mL) and gentamicin (MIC > 64 µg/mL) but remained susceptible to trimethoprim-sulfamethoxazole. Attempts to transfer resistance to β-lactams by conjugation to E. coli J53-2 with the 26 strains tested were unsuccessful. In contrast, transformants were obtained with plasmid DNA of the 19 strains tested by electroporation. The transformants' susceptibility pattern was similar to that of the donor strains, except for ciprofloxacin resistance. Analytical isoelectric focusing showed that all clinical strains and transformants had bands of β-lactamase activity with an alkaline pI of 7.6 and 5.4. Polymerase chain reaction (PCR) amplification of the 26 clinical isolates was positive for blaCTX-M and blaTEM (7). The 26 strains of E. coli had the same profile by repetitive-element PCR and pulsed-field gel electrophoresis, while unrelated control strains had very different profiles. Sequencing in strains isolated from four of the patients identified a CTX-M-15 β-lactamase and a TEM-1 β-lactamase. The four strains were related to the phylogenetic group B2 and produced the iutA (ferric aerobactin receptor), YuA (Yersinia siderophore receptor), and fimH (type I fimbriae) virulence factors (8). Incidence of colonization or infection by the culprit strain was 34.3% (12 of 35 patients) within the initial 4-month period and 55.3% (26 of 47 patients) over a 1-year period. Intensified hygienic procedures implemented in January 2002 contributed to a decrease in the number of cases in February only; since then, a regular increase of new cases extended the outbreak and caused problems with controlling it. All urinary tract infections were successfully treated with a 15-day course of trimethoprim-sulfamethoxazole; however, reinfection occurred in some. Neither incontinence (p = 0.35), dementia (p = 0.22), nor previous antibiotic treatment (amoxicillin, amoxicillin-clavulanic acid, extended-spectrum cephalosporins, and fluoroquinolones [p = 1.00, 0.30, 0.12, 0.52, respectively]) appeared to be risk factors for infection or colonization in our study, but the number of patients is too small to reach a conclusion. However, patients that were infected or colonized had greater functional impairment, especially incontinence and dementia. Nonambulatory status, decubitus ulcers, and feeding tubes were not risk factors for acquiring ESBL-producing E. coli in our study. The outbreak has not been controlled: 13 patients have persistent digestive-tract colonization. Difficulties encountered in controlling such outbreaks may be explained by several factors. Patients cannot be easily isolated in long-term care facilities. Strict isolation and limitation of activity and mobility cannot always be applied because of their impact on social activities.

Plasmid-mediated Extended-spectrum beta-Lactamases in Organisms Other Than Klebsiella pneumoniae and Escherichia coli: A Hidden Reservoir of Transferable Resistance Genes.

Resistance due to AmpC β-lactamases and ESBLs in gramnegative bacillary pathogens is a growing and important problem and is tied to extensive use of extended-spectrum cephalosporins The emergence of these β-lactamases is then amplified by spread of resistant clones or resistant genes among patients within institutions or among patients who move between institutions within a region. Genes encoding β-lactamases are frequently linked to resistance genes for other classes of antibiotics. Thus, use of any one class of antibiotic may select for emergence of resistance to another. Treatment of infections caused by these multidrug-resistant pathogens is often problematic. One strategy to circumvent β-lactamase production has been use of β-lactam/β-lactamase inhibitor combinations, but AmpC β-lactamases and hyperproduction of ESBLs evade this therapeutic strategy. Use of carbapenems and cefepime, which are the most stable of all β-lactam antibiotics to hydrolysis by ESBLs and AmpC β-lactamases, can be expected eventually to select for emergence of resistance to these drugs as well.

Plasmid-mediated extended-spectrum β-lactamase (CTX-M-3 like) from India and gene association with insertion sequence IS Ecp1

Six non-clonally related enterobacterial isolates producing a same extended-spectrum β-lactamase CTX-M-15 were isolated in 1999 from patients hospitalized in a New Delhi hospital. CTX-M-15 differed from CTX-M-3 by an asparagine to glycine substitution in position ABL238. Its gene was located on large plasmids varying in size. In each case, a same insertion sequence IS Ecp1 was identified upstream of the 5′ end of bla CTX-M-15. Typical −35 and −10 promoter sequences of Enterobacteriaceae were identified in the 3′ end of IS Ecp1. The location of IS Ecp1 upstream of plasmid-mediated CTX-M-type β-lactamase genes may contribute to their spread or/and their expression.

Plasmid-mediated resistance to expanded-spectrum cephalosporins among Enterobacter aerogenes strains.

Resistance to expanded-spectrum cephalosporins commonly develops in Enterobacter aerogenes during therapy due to selection of mutants producing high levels of the chromosomal Bush group 1 beta-lactamase. Recently, resistant strains producing plasmid-mediated extended-spectrum beta-lactamases (ESBLs) have been isolated as well. A study was designed to investigate ESBL production among 31 clinical isolates of E. aerogenes from Richmond, Va., with decreased susceptibility to expanded-spectrum cephalosporins and a positive double-disk potentiation test. Antibiotic susceptibility was determined by standard disk diffusion and agar dilution procedures. Beta-lactamases were investigated by an isoelectric focusing overlay technique which simultaneously determined isoelectric points (pIs) and substrate or inhibitor profiles. Decreased susceptibility to cefotaxime, ceftazidime, and aztreonam (MIC range, 1 to 64 microg/ml) was detected and associated with resistance to gentamicin and trimethoprim-sulfamethoxazole. All strains produced an inducible Bush group 1 beta-lactamase (pI 83). Twenty-nine of the 31 isolates also produced an enzyme similar to SHV-4 (pI 7.8), while 1 isolate each produced an enzyme similar to SHV-3 (pI 6.9) and to SHV-5 (pI 8.2). The three different SHV-derived ESBLs were transferred by transconjugation to Escherichia coli C600N and amplified by PCR. Plasmid profiles of the clinical isolates showed a variety of different large plasmids. Because of the linkage of resistance to aminoglycosides and trimethoprim-sulfamethoxazole with ESBL production, it is possible that the usage of these drugs was responsible for selecting plasmid-mediated resistance to extended-spectrum cephalosporins in E. aerogenes. Furthermore, it is important that strains such as these be recognized, because they can be responsible for institutional spread of resistance genes.

The E-Test as an epidemiologic tool

A comparison was made of the discriminating power of the E-Test and conventional methods to distinguish different bacterial strains harboring plasmid-mediated extended-spectrum β-lactamases. The E-Test provided quantitative data comparable to those of a broad range of concentrations in the agar dilution method, but proved to be far superior to the disk diffusion test or the broth microdilution semiautomatic pasco system for the detection and hence for the epidemiologic surveillance of this type of resistant strain.