Showing papers on "Aztreonam published in 2007"

An update on newer beta-lactamases.

Abstract: The resistance to beta-lactam antibiotics is an increasing problem worldwide and beta lactamases production is the most common mechanism of drug resistance. Both global and Indian figures showed a marked increase in the number of beta-lactamases producing organisms. These enzymes extended spectrum beta-lactamases (ESBLs) are numerous and continuous mutation has led to the development of enzymes having expanded substrate profile. To date, there are more than 130 TEM type and more than 50 sulphydryl variable (SHV) type beta-lactamases found in Gram negative bacilli. ESBL producing Enterobacteriaceae are, as a rule, resistant to all cephalosporins and extended spectrum penicillins including the monobactam, aztreonam, while resistance to trimethoprim - sulphamethaxazole and aminoglycosides is frequently co-transferred on the same plasmid. Many ESBL producing organisms also express Amp C beta-lactamases. Amp C- beta-lactamases are clinically significant, as these confer resistance to cephalosporins in the oxyimino group, 7 alpha-methoxy cephalosporins, and are poorly inhibited by clavulanic acid. Carbepenems are the drugs of choice for the treatment of infections caused by ESBL producing organisms but carbapenemases (MBLs) have emerged and have spread from Pseudomonas aeruginosa to Enterobacteriaceae. The routine clinical microbiology laboratories should employ simple methods to recognize these enzymes using various substrates and inhibitors. These organisms may lead to therapeutic dead ends. Presently, the therapy relies on beta-lactam/ beta-lactamases inhibitor combinations, carbepenems and piperacillin - tazobactam plus aminoglycoside combination. Proper infection control practices and barrier precautions are essential to contain the organisms producing beta-lactamases.

Plasmid-mediated carbapenem-hydrolysing β-lactamase KPC-2 in carbapenem-resistant Serratia marcescens isolates from Hangzhou, China

Abstract: Sir, Carbapenems are considered to be one of the few drugs that are useful for the treatment of infections caused by multidrugresistant isolates with high levels of AmpC or extended-spectrum b-lactamase (ESBL) production. While carbapenem resistance has become a serious problem for non-fermenting bacteria, it has remained uncommon in Enterobacteriaceae. However, identification of carbapenem-resistant Enterobacteriaceae is increasingly common. Especially, reports of nosocomial infection due to carbapenem-resistant Serratia spp. have become significantly more common, which has mainly been attributed to production of b-lactamases that are capable of hydrolysing carbapenems such as IMP-1, IMP-6, VIM-2, and SME-1, 2 and 3. The KPC class A carbapenemases are capable of hydrolysing carbapenems, penicillins, cephalosporins and aztreonam, and are inhibited by clavulanic acid and tazobactam. The first report of this b-lactamase, KPC-1, was from a carbapenemresistant Klebsiella pneumoniae. KPC-2 was then found in isolates of K. pneumoniae, Salmonella enterica, Klebsiella oxytoca and Enterobacter spp. Soon afterward, KPC-3 was found in K. pneumoniae and Enterobacter cloacae from New York. Recently, KPCs were found in France, South America and Israel. We describe three clinical isolates of carbapenem-resistant Serratia marcescens that produce KPC-2. This is the first report of detection of a plasmid-encoded carbapenem-hydrolysing enzyme KPC-2 in S. marcescens. We collected three clinical isolates of carbapenem-resistant S. marcescens from our hospital in February 2006. The three isolates exhibited either resistance or reduced susceptibility to carbapenems with imipenem MICs of 8, 64 and 8 mg/L. The isolates were also resistant to penicillins, cephalosporins and aztreonam, but were susceptible to quinolones and aminoglycosides (Table 1). Following mating of the three S. marcescens isolates with an Escherichia coli (EC600) by conjugation, all of the resulting transconjugants exhibited a phenotype of resistance to b-lactams similar to that of the S. marcescens isolate used. The imipenem and meropenem MICs of transconjugants were 1 mg/L. They were also resistant to penicillins and aztreonam, and were resistant or intermediately resistant to cephalosporins, but were susceptible to quinolones and aminoglycosides (Table 1). The three S. marcescens isolates and three transconjugants, one derived from a mating with each parent strain, had identical plasmid profiles and restriction patterns (data not shown). Following the elimination of plasmids by repeated SDS treatment, the S. marcescens isolates became susceptible to all antibiotics except ampicillin. Isoelectric focusing analysis of crude enzyme extracts from the isolates (data not shown) demonstrated the production of two b-lactamases with apparent pIs of 6.5 and 6.7. The E. coli transconjugants produced the pI 6.7 b-lactamase only. Neither pI 6.7 nor pI 6.5 b-lactamase was detected in the three plasmid-eliminated S. marcescens strains. Given the pI of 6.7, we suspected the production of a KPC b-lactamase. To confirm the presence of a blaKPC gene, we conducted PCR amplification using the primers KPC-F and KPC-R. The reaction gave a positive result and DNA sequencing identified the gene as blaKPC-2. The data show that the carbapenem-hydrolysing enzyme in the three test S. marcescens isolates is KPC-2. The enzyme is encoded on a conjugative plasmid and mainly contributed to resistance to carbapenems. We presume that the unidentified pI 6.5 b-lactamase was either the degradation product or the precursor of KPC-2. Unidentified carbapenemase activity bands around pI 6.3–6.5 have also been detected from K. pneumoniae, which were presumed to be the degradation product of KPCs. The resistance against ampicillin remaining when the blaKPC-containing plasmid had been cured is probably due to the low activity of chromosomal AmpC in S. marcescens, which was not detected in IEF but confirmed through enzyme assay. The three S. marcescens isolates had identical plasmid profiles and restriction patterns, indicating that the same plasmid spread among these isolates. Most blaKPC-encoding plasmids could be conjugated into E. coli, except the blaKPC-1-encoding plasmid described by Yigit et al. Many blaKPC-encoding plasmids also encode blaTEM-1 and various ESBLs. However, only a blaKPC gene was identified from the plasmid in S. marcescens in this study. KPC family enzymes have been detected in a variety of Enterobacteriaceae but mainly in North America. The emergence of a KPC-2 type carbapenem-hydrolysing enzyme in China is alarming. It indicates that the spread of blaKPC genes is no longer a regional problem.

Periodontal disease as reservoir for multi-resistant and hydrolytic enterobacterial species.

Abstract: Aims: This investigation aimed to isolate enteric rods from subgingival sites of patients presenting chronic periodontitis lesions, and to assess antimicrobial resistance and expression of hydrolytic enzymes. Methods and Results: Enterobacteriaceae were isolated from 20% patients, and assayed for antimicrobial susceptibility and hydrolytic enzymes with specificity to different substrates. Isolates comprised seven Enterobacter cloacae (43·75%), five Serratia marcescens (31·25%), one Klebsiella pneumoniae (6·25%), one Enterobacter aerogenes (6·25%), one Pantoea agglomerans (6·25%), and one Citrobacter freundii (6·25%). Gelatinase activity was observed for 75% strains; caseinase and elastase was produced by six and two strains, respectively. DNase, lecithinase and lipase were expressed by S. marcescens. Most of strains were resistant to ampicillin (93·75%) and amoxicillin/clavulanic acid (81·25%). The majority of strains were susceptible to cephalosporins and aztreonam. Enterobacteria remained susceptible to imipenem, streptomycin and fluoroquinolones. Resistance to gentamicin, amikacin, sulfamethoxazole/thrimethoprim, tetracycline, and chloramphenicol were also observed. Eight strains presented multiple drug resistance. Conclusions: Subgingival sites from periodontal diseases contain multi-resistant and hydrolytic enzyme-producing enterobacteria that may contribute to overall tissue destruction and spreading. Significance and Impact of the Study: Enterobacteria isolated from patients generally considered as healthy individuals poses periodontal diseases as reservoir for systemic infections particularly in immunocompromised and hospitalized hosts.

Risk factors of nosocomial infection with extended-spectrum beta-lactamase-producing bacteria in a neonatal intensive care unit in China.

Abstract: To study risk factors of neonatal nosocomial infection caused by extended-spectrum beta-lactamase (ESBL)-producing bacteria in a neonatal intensive care unit (NICU). A retrospective cohort study was conducted in a university hospital NICU in south China. Medical records of neonatal nosocomial infection caused by Escherichia coli or Klebsiella pneumoniae were reviewed. Twenty-two neonates infected with ESBL-producing bacteria (case patients) were compared with 17 patients infected with non-ESBL producing strains (controls). Univariable and multivariable logistic regression were performed to analyze risk factors for infection with ESBL-producing strains. The spectrum of antimicrobial resistance of ESBL-positive E. coli or K. pneumoniae was also examined. Both univariable and multivariable logistic regression analysis revealed that preterm low birth weight, prolonged mechanical ventilation (≥ 7 days) and prior use of third-generation cephalosporins were risks factors for ESBL-producing E. coli or K. pneumoniae infection (p 0.05). Resistance of ESBL-positive strains to piperacillin, tobramycin, aztreonam and cephalosporins was significantly higher than that of ESBL-negative ones (p < 0.05). ESBL-producing strains appeared susceptible to carbapenem, fluoroquinolones, and beta-lactamase inhibitor combination piperacillin-tazobactam. Preterm low birth weight, prolonged mechanical ventilation and prior use of third-generation cephalosporins are risks factors for nosocomial infection with ESBL-producing bacteria in NICU.

VIM-2-producing Pseudomonas putida, Buenos Aires.

Abstract: To the Editor: Pseudomonas putida (0.03% of isolates from the culture collection of the Argentina Association of Microbiology, www.aam.org.ar) infections are mainly reported in immunocompromised patients, such as newborns, neutropenic patients, and cancer patients. They are usually susceptible to extended-spectrum cephalosporins, aminoglycosides, fluoroquinolones, and carbapenems. However, isolates have been identified that produce acquired metallo-β-lactamases (MBLs) and are resistant to most β-lactams, including carbapenems. Two multidrug-resistant P. putida isolates were obtained from clinical samples at the Sanatorio Mater Dei in Buenos Aires. One isolate was obtained in March 2005 from a urine specimen of a 76-year-old woman with a urinary tract infection who was using a urethral catheter. The second isolate was obtained in May 2005 from a tracheal aspirate of a 67-year-old man with nosocomial pneumonia. Bacteria were identified by using conventional biochemical tests and the API 20NE System (API, bioMerieux, Lyon, France). Susceptibility tests were performed according to standard procedures. Both isolates were resistant to imipenem and meropenem (MICs >32 μg/mL) but were susceptible to amikacin and colistin. Susceptibility data are shown in the Table. Table Antimicrobial drug susceptibility profiles of 2 blaVIM-2-carrying Pseudomonas putida isolates, Argentina Screening for MBLs was performed by using a double-disk diffusion method. Disks containing 1 μmol/L EDTA (metal chelator) were placed on Mueller-Hinton agar plates containing the 2 isolates. Disks containing carbapenem were placed 15 mm from disks containing EDTA. An increase in the inhibition zone of the disk containing drug near the disk containing EDTA was observed for both isolates, which suggested the presence of MBLs. PCR amplification of imp and vim genes was conducted by using primers based on conserved regions of the imp and vim genes (blaIMP-F: 5′-GAAGGCGTTTATGTTCATACTT-3′, blaIMP-R: 5′-GTTTGCCTTACCATATTTGGA-3′, blaVIMG-F: 5′-GGTGTTTGGTCGCATATC-3′, and blaVIMG-R 5′-TGGGCCATTCAGCCAGATC-3′) and heat-extracted DNA as template. Reactions were performed in a T-gradient instrument (Biometra, Gottingen, Germany) with the following reaction conditions: 1 cycle at 95°C for 5 min, 52°C for 15 min, and 72°C for 6 min, followed by 30 cycles at 95°C for 1 min, 52°C for 1 min, and 72°C for 1 min, and a final reaction at 72°C for 20 min. Amplified fragments were sequenced on both strands by using an ABI Prism DNA 3700 (Applied Biosystems, Foster City, CA, USA), and nucleotide sequences were compared by using BLAST (National Center for Biotechnology Information, Bethesda, MD, USA, http://www.ncbi.nlm.nih.gov/Tools/). Nucleotide sequences were completely homologous to the vim-2 coding gene. Two repetitive-element–based PCR (rep-PCR) assays (ERIC-PCR and REP-PCR) with primers REP-1 (5′-IGCGCCGICATCAGGC-3′), REP-2 (5′-CGTCTTATCAGGCCTAC-3′), ERIC-1 (5′-CACTTAGGGGTCCTCAATGTA-3′), and ERIC-2 (5′-AAGTAAGTGACTGGGGTGAGCG-3′) were used to characterize isolates. PCR conditions were 94°C for 2 min, 30 cycles at 94°C for 30 s, 50°C for 1 min, and 72°C for 4 min, and a final reaction at 72°C for 7 min. Banding patterns were visually analyzed after electrophoresis of samples. Variations in band intensity were not considered to indicate genetic differences. Banding patterns obtained by REP-PCR and ERIC-PCR assays were identical in both isolates (data not shown). Among the MBLs acquired by P. putida, IMP-1 was reported by Senda et al. in Japan in 1996 (1) and later reported in Taiwan and Japan (2). IMP-12 was the first IMP MBL described in P. putida in Europe (3). VIM-1 in P. putida was first reported in Europe (4), and VIM-2 in P. putida was first reported in Taiwan, Republic of Korea, Japan, and France (5,6). Our isolates were resistant to aztreonam (MIC 64 μg/mL). However, carbapenem-susceptible P. putida had low levels of susceptibility because the MIC50 was only 1 dilution below the current breakpoint (7,8). Aztreonam resistance could not be transferred by conjugation between IMP-1–producing (aztreonam-resistant) P. putida and P. aeruginosa (2) and is not associated with a transposon carrying blaVIM-2 (6). No evidence of extended-spectrum β-lactamases was detected in our isolates by classic synergy assays with clavulanate plus aztreonam, ceftazidime, or cefotaxime. VIM-6–producing P. putida isolates from Singapore (9) were more resistant to aztreonam (MIC >128 μg/mL), ceftazidime, and cefepime (MIC >256 μg/mL). Detection of blaVIM-2 in Pseudomonas in South America was initially reported by the SENTRY Antimicrobial Surveillance Program (10) and included 1 P. fluorescens isolate in Chile and 3 P. aeruginosa isolates in Venezuela. To the best of our knowledge, our report is the first of VIM-2 in P. putida in Latin America. VIM-2–producing P. putida, which were originally restricted to East Asia and only very recently found in France, may represent an emerging pathogen or function as reservoirs for resistance because of their widespread presence in the hospital environment.

Pseudomonas aeruginosa: freqüência de resistência a múltiplos fármacos e resistência cruzada entre antimicrobianos no Recife/PE

Abstract: BACKGROUND AND OBJECTIVES: The frequency of multiple-antibiotic resistant bacteria has been increasing in recent years. Among the gram-negative bacteria Pseudomonas aeruginosa (P. aeruginosa) shows a great propensity for the development of multidrug resistance mechanisms. The objective of this study was to identify the profile of susceptibility to antibiotics, the frequency of multidrug resistance and the cross-resistance between drugs of P. aeruginosa strains in two tertiary hospitals in Recife, Pernambuco. METHODS: The study was carried out between September 2004 and January 2006. The antimicrobial susceptibility testing was performed in 304 strains of P. aeruginosa by the disc diffusion method in accordance with National Committee for Clinical and Laboratory Standards (NCCLS) guidelines. RESULTS: The most frequent materials were urine (26.7%) and respiratory tract secretion (26.1%) The antibiotics tested and their respective susceptibilities were as follows: piperacillin-tazobactam (66.2%); aztreonam (59.8%); amikacin (59.4%); meropenem (58.2%); imipenem (57.7%); ciprofloxacin (49.7%); gentamicin and cefepime (48.6%); ceftazidime (30%) and cefotaxime (6.8%). A high prevalence of multi-resistance was detected. Half (49.7%) the strains showed resistance to three or more antibiotics and 28% were resistant to six antimicrobials or more. Also, cross-resistance between the beta-lactams (carbapenems and piperacilin/tazobactam) and aminoglicosides and quinolones was between 22.9% and 38.1%. These drugs are commonly combined in the treatment of severe infections caused by Pseudomonas, which reflects the difficulty in choosing the appropriate option for combination therapy. CONCLUSIONS: The frequency of multidrug-resistant strains of P. aeruginosa in this study was similar to other hospitals in Brazil and higher than in other countries. In order to reduce the frequency of these multiresistant clones, epidemiologic surveillance and the rational use of antibiotic protocols need to be urgently implemented.

First detection and sequence analysis of the bla-CTX-M-15 gene in Lebanese isolates of extended-spectrum-β-lactamase-producing Shigella sonnei

Abstract: The emergence in Shigella species of extended-spectrum beta-lactamases (ESBL) that impart resistance to third-generation cephalosporins is a growing concern world-wide. So far, however, ESBL-producing Shigella have only been reported seven times, albeit from seven different countries. In Lebanon, three ESBL-producing clinical isolates of S. sonnei were recovered from 30 cases of shigellosis diagnosed between July 2004 and October 2005. All three were found to be resistant to amoxycillin, cefotaxime, ceftazidime, aztreonam, trimethoprim/sulphamethoxazole, gentamicin, and kanamycin. Each harboured the bla-CTX-M gene, and the results of sequence analysis indicated this to be of the bla-CTX-M-15 type and encoded on a 70-kb plasmid, flanked by an insertion element (ISEcp1). The bla-TEM-1 gene was also detected on the chromosomes of two of the ESBL-producing isolates. Class-2 integrons containing dhfr1, aadA1 and sat1 genes were detected on the chromosomes of all three isolates but not on the plasmids. Fluoroquinolone-modifying factors [QnrA, QnrB, QnrS or AAC(6')-Ib-cr] were not detected. The results of RAPD analysis, combined with data on antimicrobial susceptibility, indicated that each isolate was unique. In conclusion, the emergence of ESBL-producing isolates of S. sonnei has been demonstrated for the first time in Lebanon. The resistance of these isolates to third-generation cephalosporins was mediated by the CTX-M-15 enzyme, which was plasmid-encoded.

Prevalence of extended-spectrum beta-lactamases-producing isolates over a 1-year period at a University Hospital in Oman.

Abstract: OBJECTIVES To evaluate the prevalence of extended-spectrum beta-lactamases isolates over one year period at Sultan Qaboos University Hospital. METHODS We identified the ESBL isolates during a 12-month period from July 2004 to June 2005, using a commercial system, and confirmed the result using the National Committee for Clinical Laboratory Standards-approved double-disk diffusion method. RESULTS Sensitivity was recorded for a wide range of antibiotics, aminoglycosides, carbapenem, cephalosporins, quinolones, aztreonam, ampicillin, amoxicillin/clavulanate, ampicillin/sulbactam, piperacillin-tazobactam, trimethoprim/ sulfamethoxazole and nitrofurantoin. Of the total ESBL isolated, 29.6% were from medical ward, followed by outpatients clinic, 24.3%. Urine was the main source of ESBLs 70.4%, followed by 16.5% from blood. We observed a 100% sensitivity to carbapenems, whereas 93.9% of the isolates were susceptible to amikacin. Cephalosporins were 100% resistant, except for cefoxitin, which demonstrated sensitivity of 77.4%. Aztreonam, ampicillin, co-amoxyclav and ampicillin/sulbactam were 100% resistant. Of the isolates, 57.4% were sensitive to nitrofurantoin, whereas Tazocin showed 49.6% sensitivity and co-trimoxazole 13.9%. To quinolones, 74.8% of the isolates were resistant. CONCLUSIONS Excess use of third generation cephalosporins led to increase rate of ESBLs, which are difficult to treat. Carbapenem are most reliable for treatment of infections caused by ESBL isolates. However, overuse of carbapenem may lead to resistance of other gram-negative organisms. Therefore, justifiable use of third-generation cephalosporins, will be an effective means of controlling and decreasing the spread of ESBL isolates.

Role of β-lactamase inhibitors in enterobacterial isolates producing extended-spectrum β-lactamases

Abstract: OBJECTIVES: To determine the in vitro activity of beta-lactamase inhibitors (clavulanic acid and sulbactam) in combination with third-generation cephalosporins and monobactam against extended-spectrum beta-lactamase (ESBL)-producing members of the Enterobacteriaceae family. METHODS: A total of 361 ESBL-producing enterobacterial isolates obtained from patients of a university hospital were screened for the status of co-production of AmpC beta-lactamase. These strains were further subjected to an MIC study using third-generation cephalosporins and monobactam, and reductions were observed after combining with beta-lactamase inhibitors at a fixed concentration of 4 mg/L. RESULTS: Most of the isolates showed 8-fold reduction with sulbactam when combined with ceftriaxone, cefpodoxime and cefotaxime but not with ceftazidime and aztreonam, whereas clavulanic acid showed the same result with all the cephalosporins tested. Further, both the inhibitors showed greater reduced MIC when combined with aztreonam. CONCLUSIONS: As the ability of clavulanic acid to induce AmpC production may interfere with ESBL detection, sulbactam is likely to be preferred over clavulanic acid after standardization of an appropriate concentration for ESBL detection in the scenario of increased prevalence of AmpC producers. Greater in vitro activity of these inhibitors when combined with aztreonam further indicates the need of studies to evaluate these combination antimicrobials in clinical settings as they can play a significant role for clinicians as viable alternatives to treat infections caused by such organisms.

Prevalence of fecal carriage of ESBL-producing Enterobacteriaceae in hospitalized and ambulatory patients during two non-outbreak periods.

Abstract: Beta-lactam resistance in Enterobacteriaceae associated with plasmid-mediated extended-spectrum beta-lactamase (ESBL) has become a worldwide problem [1]. ESBLs were initially associated with nosocomial outbreaks caused by strains producing single enzymes, but recent studies have revealed a more complex situation, with a significant increase among community isolates [2]. The rate of fecal carriage of ESBL-producing bacterial isolates in nosocomial outbreaks has been studied previously [3, 4], but few prospective studies investigating the prevalence of fecal carriage during non-outbreak periods have been conducted [5, 6]. In an effort to fill this knowledge gap, we performed a study of hospitalized patients and outpatients to determine the prevalence of fecal carriage of ESBL-producing Enterobacteriaceae in stool samples submitted to our laboratory for culture during two non-outbreak periods separated by 17 months (April 2002–February 2003 and August 2004– July 2005). A total of 7,065 and 7,686 fecal samples from 4,676 and 4,708 patients were analyzed in each period, respectively. Ambulatory patients accounted for 75.5 and 70% of the total in each respective period. The samples were cultured using standard methods for Salmonella, Shigella, Campylobacter, Yersinia, Aeromonas and Plesiomonas. In 13.8% of all stool cultures collected during both periods, other nonCampylobacter strains belonging to the family Enterobacteriaceae were isolated. These grew in modified charcoal-cefazolin-deoxycholate agar containing cefoperazone 32 mg/l and amphotericin B supplement (Oxoid, Basingstoke, UK) incubated for 48 h under microaerobic conditions—a method used routinely to isolate Campylobacter spp in our laboratory. All of the non-Campylobacter strains that grew in the medium containing cefoperazone were identified according to conventional methods [7] and screened for production of ESBL by identification of the resistance phenotype and using the double-disk synergy test with cefotaxime, ceftazidime, cefepime, aztreonam and amoxicillin-clavulanic acid [8]. Results were confirmed by determining the MICs using the E-test (AB Biodisk, Solna, Sweden) with cefotaxime/cefotaxime-clavulanic acid, ceftazidime/ceftazidime-clavulanic acid and cefepime/cefepime-clavulanic acid. Strains producing ESBL were defined as strains showing synergism between clavulanic acid and cefotaxime, ceftazidime, cefepime and/or aztreonam [9]. All strains suspected of having a resistance pattern compatible with hyperproduction of the chromosomal enzymes as well as resistant strains without synergy were excluded from the study. Of the enterobacteria isolated in the modified charcoalcefazolin-deoxycholate agar, 16.3 and 26.76% (105 of 643 and 349 of 1,304 in each period, respectively) were ESBLproducing strains. Statistical significance was calculated for comparison of proportions using the chi-square test, and a p value of <0.05 was considered statistically significant (SPSS V 12.0; SPSS, Chicago, IL, USA). The rates of fecal carriage of ESBL-producing strains belonging to the Enterobacteriaceae family increased dramatically in our area from 2.3% (105 of 4,676 patients) in 2002 to 7.4% (349 of 4,708 patients) in 2004 (p<0.001). Eur J Clin Microbiol Infect Dis (2007) 26:77–78 DOI 10.1007/s10096-006-0242-8

Evolution of the antimicrobial resistance of Pseudomonas aeruginosa in Spain: Second National Study (2003)

Abstract: The second national prevalence study of Pseudomonas aeruginosa has been carried out in Spain. A total of 1250 clinical isolates of P. aeruginosa were collected from 127 hospitals in 1 week in 2003 and the resistance data gathered from the isolates was compared with those of the first study in 1998 (1014 isolates from 136 hospitals). Antimicrobial susceptibility testing was performed in both studies in the same laboratory. The most active antimicrobials were piperacillin, piperacillin-tazobactam, and amikacin (≤10% resistant) and resistance to these antimicrobials did not change over the time. The least active were ofloxacin and gentamicin (≥30% resistant). From 1998 to 2003, resistance increased significantly to ciprofloxacin (23% vs. 28%, respectively, p = 0.015); ofloxacin (30% vs. 37%, p = 0.002); imipenem (14% vs. 18%, p = 0.017) and meropenem (8% vs. 13%, p <0.001). Resistance to aztreonam (23%), ceftazidime (16%), cefepime (20%), ticarcillin (13%) and tobramycin (11%) remained stable. Isolates from inpatients were significantly more resistant than those from outpatients to all antimicrobials, with the exception of fluoroquinolones and aminoglycosides (p <0.01). Isolates from outpatients were significantly more resistant to these two groups (p <0.05) than to other antimicrobials. In Spain, from 1998 to 2003, the susceptibility pattern of P. aeruginosa to antimicrobial agents has changed. Isolates have become significantly more resistant to fluoroquinolones and carbapenems. However, resistance to beta-lactams and aminoglycosides remains stable. © 2007 Prous Science, S.A. Sociedad Espanola de Quimioterapia.

Biochemical Characterization of SFC-1, a Class A Carbapenem-Hydrolyzing β-Lactamase

Abstract: The carbapenem-hydrolyzing β-lactamase SFC-1 from Serratia fonticola UTAD54 was overexpressed in Escherichia coli, purified, and characterized. The enzyme exhibited an apparent molecular mass of 30.5 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. SFC-1 hydrolyzes penicillins, cephalosporins, aztreonam, and carbapenems and is inhibited by clavulanic acid, sulbactam, and tazobactam.

SME-2-Producing Serratia marcescens Isolate from Switzerland

Abstract: Carbapenemases in Serratia marcescens have been rarely reported, being related either to metallo-β-lactamases IMP-1, IMP-6, and VIM-2 (10) or to SME-type Ambler class A β-lactamases (6). SME-1 had been identified first from the carbapenem-resistant S. marcescens strain S6, isolated in London in 1982 (5), and then in carbapenem-resistant S. marcescens strains isolated in 1999 in the United States (1). It hydrolyzes penicillins, aztreonam, cephalosporins, and carbapenems and is inhibited by clavulanic acid (4). The blaSME-1 gene was chromosome encoded in isolate S6 (5). The SME-2 and SME-3 variants have been identified (being just point mutant analogues) from S. marcescens isolates recovered from the United States and United Kingdom (8, 9). Our study was initiated by the isolation in May 2006 at the University Hospital of Lausanne, Switzerland, of a carbapenem-resistant S. marcescens isolate. This isolate was from an exudate obtained after a parotidectomy of a 62-year-old patient who received a prophylaxis containing amoxicillin-clavulanate. Three days after surgery, the patient suffered from a pulmonary deficiency and received an imipenem-containing treatment. Two days later, culture of the exudate gave S. marcescens strain AW, which was resistant to penicillins and imipenem. The MICs of imipenem, meropenem, and ertapenem were 32, 8, and 4 μg/ml, respectively. It was also resistant to cefoxitin and aztreonam, whereas it remained susceptible in vitro to expanded-spectrum cephalosporins. The results for double-disk synergy testing, performed as described previously (2), were slightly positive with the clavulanate-imipenem combinations, indicating that S. marcescens AW produced a carbapenemase that was inhibited by clavulanic acid. PCR amplification using various primers, including blaSME-specific primers and whole-cell DNA of S. marcescens AW, followed by sequencing performed as described previously (7), identified a gene encoding SME-2. No other similar isolate was recovered from the same hospital during the same period of time, and no history of travel abroad or previous hospitalization was identified. The clonal relationship between isolate AW and isolate S6 from London was evaluated by pulsed-field gel electrophoresis as described previously (8), showing that isolate AW was not clonally related to isolate S6. By analyzing the sequences of the rpoB genes as reported previously (3), we found that the sequences obtained from isolates AW and S6 and also from two randomly selected carbapenem-susceptible and blaSME-negative S. marcescens isolates were identical. This observation likely rules out a possible identity for SME producers as part of a given subspecies. Conjugation, electrotransformation, and plasmid analysis, performed as described previously (5, 7), failed to identify a plasmid-borne location for the blaSME-2 gene, suggesting a likely chromosomal location for this gene. Further PCR mapping showed the presence of the LysR-type regulatory gene smeR upstream of blaSME-2, as previously identified (4). This was in accordance with the observation of a slight antagonism observed between the cefoxitin- and imipenem-containing disks. This report underlines the possible identification of SME-type S. marcescens producers worldwide. Along with other clavulanic acid-inhibited carbapenemases (NMC-A, IMI, KPC), SME-type enzymes may confer clinically significant resistance to carbapenems. Interestingly, SME producers may confer a lower level of resistance to ertapenem than to the other carbapenems.

Extended-spectrum beta-lactamase gram-negative sepsis following prostate biopsy: implications for use of fluoroquinolone prophylaxis.

Abstract: Extended-spectrum beta-lactamase (ESBL) producing organisms are resistant to penicillins, cephalosporins, aminoglycosides, trimethoprim-sulfamethoxazole, aztreonam, and most fluoroquinolones. We report a case of a 72-year old man who developed septic shock with an ESBL organism after a transrectal ultrasound (TRUS)-guided prostate biopsy despite having received fluoroquinolone prophylaxis. The patient recovered with intravenous ertapenem. Fluoroquinolone resistant bacteria are increasing in prevalence. This needs to be recognized when the antibiotic choice for pre-procedure prophylaxis is made.

Widespread detection of VEB-1-type extended-spectrum beta-lactamases among nosocomial ceftazidime-resistant Pseudomonas aeruginosa isolates in Sofia, Bulgaria.

Abstract: A total of 132 ceftazidime-resistant clinical isolates of Pseudomonas aeruginosa were collected during 2001-2005 from 5 university hospitals in Sofia, Bulgaria to assess the current levels of antimicrobial susceptibility and to evaluate resistance mechanisms to beta-lactams. Antimicrobial susceptibilities were detected by a disk diffusion method and E-test. Polymerase chain reaction amplification and sequencing of bla(VEB-1 )and bla(PER-1 )were performed. The antibiotic resistance rates were: to piperacillin 90.2%, piperacillin/tazobactam 52.3%, ceftazidime 94.7%, cefepime 88.6%, cefpirome 98.5%, aztreonam 85.6%, imipenem 66.6%, meropenem 63.6%, amikacin 81.1%, gentamicin 84.8%, tobramycin 89.4%, netilmicin 57.6%, ciprofloxacin 83.4%. Structural genes for VEB-1 extended-spectrum beta -lactamases (ESBLs) were found in 75 (56.8%) of the isolates. PER-1 ESBLs were not detected. The VEB-1-producing strains were more resistant than VEB-1 non-producers to amikacin, gentamicin, tobramycin and ciprofloxacin ( P<0.001). VEB-1 appears to have a significant presence among ceftazidime-resistant P. aeruginosa isolates from Sofia.

Providencia stuartii with VIM-1 metallo-β-lactamase

Abstract: Sir, Providencia stuartii is a frequent cause of urinary tract infections in hospitalized and nursing home patients with long-term indwelling urinary catheters. Also, this opportunistic pathogen is occasionally implicated in other types of hospital-acquired infections. Clinical strains of P. stuartii are commonly resistant to aminopenicillins and early generation cephalosporins due to production of an inherent cephalosporinase. Furthermore, they can acquire plasmids encoding class A extended-spectrum b-lactamases (ESBLs) such as TEM, CTX-M and VEB conferring resistance to newer cephalosporins. – 3 In this study, a ceftazidime-resistant strain of P. stuartii producing VIM-1 metallo-b-lactamase (MBL) is described. blaVIM-1 was carried by a self-transferable plasmid. P. stuartii 323/07 was derived from a patient in the ICU of ‘Eugenidion’ General Hospital in Athens. The patient (male, 72 years old) was intubated due to severe ischaemic stroke of the left brain hemisphere and was requiring mechanical ventilation since 10 October 2006. He was transferred to our ICU on 19 October with a lung infection due to aspiration pneumonia. After being stabilized, a new septic event with fever, purulent secretions and haemodynamic instability emerged on 19 January 2007, while on treatment with meropenem and linezolid. Cultures of blood and bronchial secretions yielded both Pseudomonas aeruginosa and Acinetobacter baumannii, and antimicrobial treatment was switched to ceftazidime and colistin with no clinical improvement. A multiresistant but ciprofloxacin-susceptible strain of P. stuartii was isolated from bronchial secretions on 29 January and colistin was replaced by ciprofloxacin. The latter regimen was apparently effective, resulting in a significant clinical improvement within 1 week. MICs of b-lactams were determined by an agar dilution technique. Susceptibility status to non-b-lactams drugs was assessed by a disc diffusion method. P. stuartii 323/07 exhibited resistance or decreased susceptibility to penicillins, penicillin/ inhibitor combinations, ceftazidime and cefotaxime. MICs of carbapenems, aztreonam and cefepime were within the susceptibility range (Table 1). The strain was also resistant to gentamicin, tobramycin, netilmicin, co-trimoxazole, tetracycline and chloramphenicol. Amikacin and fluorinated quinolones were active against P. stuartii 323/07. The strain was negative for ESBL production in the double disc synergy test. Also, addition of either Ro 48-1220 or clavulanic acid did not reduce the MIC of ceftazidime (Table 1), indicating that the observed phenotype was not due to production of an ESBL or a cephalosporinase. However, P. stuartii 323/07 was positive in the double disc test based on synergy between imipenem and EDTA. PCR assays using total bacterial DNA as template and primers specific for blaVIM-1 were positive. Isoelectric focusing of b-lactamases showed that P. stuartii 323/ 07 produced an enzyme with a pI of 5.1, similar to that of VIM-1. A b-lactamase with an apparent pI of 8.5 was also detected. This was probably the chromosomal AmpC of P. stuartii. Conjugal transfer of resistance to b-lactam antibiotics to an Escherichia coli K12 recipient strain highly resistant to rifampicin was carried out in mixed broth cultures and selection on media containing rifampicin and ampicillin. Resistance to b-lactams as well as aminoglycosides and co-trimoxazole was readily transferred at a frequency of 10 transconjugants per donor cell. E. coli transconjugants were positive for blaVIM-1 by PCR. Agarose gel electrophoresis of plasmid DNA preparations from P. stuartii 323/07 and an E. coli transconjugant indicated transfer of a single plasmid (pPS-v1) of 60 kb that was positive for blaVIM-1 in hybridization experiments performed as described previously. PCR mapping was carried out using combinations of oligonucleotide primers specific for the 50 and 30 conserved sequences of class 1 integrons as well as blaVIM-1 and various resistance genes. These experiments showed that blaVIM-1 was the first gene cassette of an integron that also contained—from 50 to 30—aacA4, dhfrI, aadA and sulI, similar to In-e541 described previously in Klebsiella pneumoniae and E. coli (GenBank accession no. AY339625). pPS-v1 belonged to the incompatibility (Inc) group N as determined by a PCR-based Inc-typing method. It also exhibited a PstI-generated restriction profile similar to that of a group of IncN blaVIM-1carrying plasmids that have been spread mainly among K. pneumoniae (data not shown). The higher resistance levels to various b-lactams, including imipenem, of the E. coli transconjugant clones when compared with P. stuartii 323/07 (Table 1) suggested differences in the production of VIM-1. To validate this hypothesis, hydrolytic activities of bacterial cell extracts against imipenem were determined by spectrophotometry. Extracts from E. coli (pPS-v1) expressed approximately 5-fold higher activity than P. stuartii 323/07 that was consistent with the b-lactam MIC differences. Additionally, transconjugants were resistant to amikacin, whereas P. stuartii 323/07 appeared susceptible to this drug, probably indicating differences also in the expression of the aacA4 gene cassette. To the best of our knowledge, this is the first description of a P. stuartii strain producing a VIM-type MBL. This finding underscores the ongoing spread of VIM-1 in the flora of Greek hospitals as well as the important role of IncN, blaVIM-1-carrying plasmids in this process. Journal of Antimicrobial Chemotherapy doi:10.1093/jac/dkm139

Sensitivity and specificity of various β-lactam antibiotics and phenotypical methods for detection of TEM, SHV and CTX-M extended-spectrum β-lactamases

Abstract: The aim of this study was to compare the sensitivity and specificity of six different beta-lactam antibiotics using five phenotypical tests for detection of extended spectrum beta-lactamases (ESBLs) based on synergism of beta-lactam antibiotics and clavulanate. Experiments were performed on a set of 80 Klebsiella pneumoniae strains and 105 Escherichia coli strains with previously characterized ESBLs (SHV, TEM and CTX-M). ESBLs were detected by five different phenotypical methods: MIC (minimum inhibitory concentration) determination of beta-lactam antibiotics with and without clavulanate, double-disk synergy test (DDST), inhibitor-potentiated disk-diffusion test (IPDDT), CLSI-Clinical and Laboratory Standard Institution (former NCCLS) combined-disk-test, and modified MAST-disk-diffusion test (MAST-DD-test). Seven antibiotics were tested as indicators of ESBL production: ceftazidime, cefotaxime, ceftriaxone, aztreonam, ceftibuten, cefpodoxime and cefepime. Ceftazidime and aztreonam were the best indicators for SHV-5, SHV-12 and TEM beta-lactamases whereas cefotaxime and ceftriaxone were the most sensitive in detection of SHV-2 and CTX-M beta-lactamases in DDST, IPDDT and CLSI test. MIC determination of beta-lactam antibiotics with and without clavulanate was the most sensitive method. DDST was the least sensitive test. Double-disk synergy test, which is the most frequently used test for detection of ESBLs in routine laboratories, was the least sensitive independently of the indicator antibiotic. Since MIC determination is a very laborious and time consuming method, we would recommend the NCCLS combined disk test or IPDD test for detection of ESBLs in routine laboratories with 5 mm zone augmentation breakpoint.

Caractérisation des beta-lactamases et leur inhibition par les extraits de plantes médicinales

Abstract: J. Gangoue Pieboji These de Doctorat xi ABSTRACT Bacteria producing extended-spectrum β-lactamase (ESBL) have been reported in many countries, but there is no information on different type of ESBL-producing enterobacteria in Cameroon. More than 290 β-lactamases have been described and divided into four classes (A, B, C and D) and up to now, there is no good inhibitor which can inhibite all classes of β-lactamases. A total of 267 strains of Enterobacteriaceaae were isolated between 1995-1998 (259 isolates) and 2002 (eight isolates) from pathological products (urines, pus and blood) of patients at the Yaounde Central Hospital. The susceptibility of the isolates to 12 antibiotics (amoxicillin, amoxicillin/clavulanate, piperacillin, imipenem, cefazolin, cefoxitin, cefotaxime, ceftazidime, aztreonam, gentamicin, ofloxacin and trimethoprim/sulfamethoxazole) was determined using the agar diffusion disk method. Imipenem, ofloxacin and ceftazidime were the most active antibiotics against overall enterobacteria with susceptibility rates of 100%, 91% and 88% respectively. High resistance rates were observed to amoxicillin (90%), piperacillin (79%), cefazolin (75%) and trimethoprim/sulfamethoxazole (73%). Enterobacteria isolates (69) resistant to oxyimino-cephalosporin or monobactam were screened for ESBL production by the double disk (DD) synergy test. Thirty-eight (31 from 1995-1998; seven in 2002) were proved positive to the DD synergy test, suggesting the presence of ESBL. The prevalence of ESBL was 12% (31/259) and ESBL-producing strains were Klebsiella spp. (18.8%), Citrobacter spp. (17.6%), Escherichia coli (14.3%) and Proteus spp. (1.8%). Of the 38 ESBL-producing strains, 18 (47.4%) were transferred resistance to oxyimino-cephalosporins to E. coli HK 225, K12 and DH5α. Sixteen of these strains were transferred ESBL gene by conjugation and two by transformation. Resistance to gentamicin and/or trimethoprim/sulfamethoxazole was co-transferred. All transconjugant and transformant strains exhibited ESBL phenotype but remained susceptible to cefoxitin and imipenem. Crude extracts of β-lactamase-producing transconjugants, transformants and clinical strains were able to reduce the diameters of inhibition zones around disks containing penicillins, 1 to 3 generation cephalosporins or monobactam when tested against a fully susceptible E. coli strains, but had no effect on such zones around cefoxitin-, imipenemand amoxicillin/clavulanate disks. The determination of isoelectric points (pIs) of the various strains after transfer of resistance determinant showed that transconjugants and transformants Caracterisation et inhibition des beta-lactamases par les plantes medicinales These de Doctorat xii strains produced β-lactamases with pIs 5.4 to 8.8. In fact, 12 strains produced β-lactamase with pI 8.2, 2 strains in addition to the above enzyme produced two others enzymes with pIs 5.4 and 8.5. Strains with CTX-Mphenotype produced enzyme (pI 8.4, 1 strain), two (pIs 7.3, 8.8; 2 strains) or three types pIs (5.4, 7.3, 8.8; 1 strain). PCR, PCR/NheI was performed using total or plasmid DNA from 19 strains as templates, and the primers specific to blaSHV, blaCTX-M, blaTEM and blaOXA. Direct sequencing of PCR products revealed that: 14 strains produced SHV-12 ESBL, 5 CTX-MESBL (CTXM-1, one strain; CTX-M-15, 4 strains); four other strains shared non-ESBL TEM-1 (2 producing-SHV-12 strains and 2 producing-CTX-M-strains) and OXA-30 (4 producing CTXM-15 strains). Analysis of restriction patterns of plasmid carrying blaCTX-M gene, hybridization, and genotyping patterns of CTX-M-clinical strains by ERIC-PCR showed that dissemination of blaCTX-M gene could be done by plasmid transfer (same plasmid in different strains, E. coli YC-14 and K. pneumoniae YC-17) or by strains from patient to patient (same strain, E. coli YC-2=E. coli YC-5). Sequence analysis of genetic environment of blaCTX-M-15 revealed that CTXM-15 was encoded by two different multiresistant plasmids, of which one (pYC-5b) carried an ISEcp1-blaCTX-m-15 element flanked by five bp target site duplication and inserted within a Tn2-derived sequence. A truncated form of this element in the second plasmid (pYC14) was identified. ESBLs SHV-12, CTX-M-1, CTX-M-15 are described for the first time in Cameroon. In our effort to find new active β-lactamase inhibitors, we investigated two medicinal plants, Garcinia lucida (Clusiaceae) and Bridelia micrantha (Euphorbiaceae) for antiβlactamase activity. The extracts from G. lucida and B. micrantha exhibited good inhibition of β-lactamase P99 (IC50, 0.01 mg/ml) and OXA-10 (IC50, 0.02 mg/ml) respectively. Purified products from these plants by high performance liquid chromatography showed that, product 4 from G. lucida is very active on β-lactamase P99 (IC50, 0.03 mg/ml) whereas products 2’ and 3’ from B. micrantha are active on OXA-10 (IC50, 0.09 mg/ml; 0.11 mg/ml respectively). The structural elucidation of the active constituents of these plants will provide useful leads in the development of β-lactamase inhibitors.

Tobramycin-Induced Hepatotoxicity

Abstract: Objective:To report a case of tobramycin-induced hepatotoxicity.Case Summary:A 20-year-old female was hospitalized for treatment of Pseudomonas aeruginosa bacteremia and osteomyelitis. Empiric intravenous antibiotic therapy with piperacillin/tazobactam, vancomycin, and ciprofloxacin was started, and based on the results of culture and sensitivity testing, was changed to intravenous ceftazidime and tobramycin 70 mg every 8 hours on hospital day 3. Liver enzyme levels then increased over days 3–6. Tests for hepatitis A, B, and C were all nonreactive, and HIV testing was negative. On day 8, therapy was changed from ceftazidime to piperacillin/tazobactam and the tobramycin dose was increased to 100 mg every 8 hours. Due to a continued increase in total bilirubin, aspartate aminotransferase, and alanine aminotransferase, piperacillin/tazobactam was discontinued and aztreonam was started on day 10. All antibiotics were stopped on day 12 and the elevated liver parameters began to decrease. Aztreonam and ciproflo...

A study of clinical strains of Pseudomonas aeruginosa and the investigation of antibiotic resistance mechanisms in the multidrug resistant strain PA13

Abstract: Thirteen clinical strains of bacteria from two Irish hospitals were identified as Pseudomonas aeruginosa using classical methods, API 20NE and Biolog GN. Their identification was confirmed by 16S rRNA gene sequencing. The antibiotic resistance profiles of the isolates were determined against forty-one antibiotics belonging to eleven distinct classes. All the isolates were resistant to penicillin G, ampicillin, cephalothin, cloxacillin, oxacillin, amoxicillin, cefotaxime, moxalactam, sulphatriad cotrimoxazole, chloramphenicol and tetracycline. All were sensitive to ceftazidime, piperacillin-tazobactam, cefepime, ceftriaxone, meropenem, aztreonam, amikacin, apramycin, butirosin A, lividomycin and colistin sulphate. One of the isolates, PA13, was resistant to a further fourteen antibiotics and was identified as a multidrug resistant strain. A 2.2 kbp PCR product was amplified from P. aeruginosa PA13. When this product was sequenced it was found to contain four open reading frames. BLASTN analysis identified these as being an integrase gene (ORF1), an aminoglycoside acetyltransferase gene, aac(6’)-Ib (ORF2), an oxacillinase gene (ORF3) and a quaternary ammonium compound resistance gene (ORF4). The presence of the integrase gene and the quaternary ammonium compound gene suggested that the genes were on a Class 1 integron. The acetyltransferase aac(6’)-Ib gene contained the mutant type of the enzyme with a leucine substitution by serine at position 119. Two expression vectors were chosen to investigate the novel oxacillinase gene. One was a commercially available vector, pET-28a (Novagen) and the other was an in-house vector, pPC. The gene was successfully cloned into both vectors. Following induction the desired protein was not expressed in either the soluble or insoluble fractions.

CHINET 2005 surveillance of antimicrobial resistance in Pseudomonas aeruginosa in China

Abstract: Objective To investigate the resistance of clinical isolates of Paeruginosa in China during 2005Methods Clinical isolates of Paeruginosa from 7 teaching hospitals in China were collected and antimicrobial susceptibility were tested by Kirby-Bauer methodResults A total of 2 323 clinical isolates of Paeruginosa were collectedThe resistance rates of Paeruginosa to antimicrobial agents from low to high were amikacin(225%),cefoperazone-sulbactam(228%),cefepime(276%),ceftazidime(289%),imipenem(313%),ciprofloxacin(317%),meropenem(337%),piperacillin-tazobactam(344%),aztreonam(368%),piperacillin(442%),ticarcillin-clavulanic acid(509%)Multi-drug resistant(MDR)and pan-drug resistant(PDR)strains accounted for 84% and 42%,respectivelySome(367%-534%)of the strains resistant to other antimicrobial agents were still susceptible to amikacinTherefore,combination therapy of β-lactams plus an aminoglycoside is usually indicated for serious infections caused by PaeruginosaBy 'interpretative reading' of the susceptibility pattern of 'predictive drugs',we can predict the underlying resistance mechanismsConclusions The surveillance data indicate that the resistance of Paeruginosa in China is relatively serious and its resistance mechanisms are very complicatedClose attention should be paid to this problem by physicians and clinical laboratory

284 CARBAPENEM-RESISTANT STRAIN OF KLEBSIELLA PNEUMONIAE HARBORING ONE COPY OF THE SERINE CARBAPENEMASE KPC-2 AND TWO COPIES OF A TEM-TYPE EXTENDED-SPECTRUM β-LACTAMASE GENE.

Abstract: Background Strains of Klebsiella pneumoniae with multiple determinants of drug resistance present a serious management problem. The determinants that confer resistance to cephalosporins and carbapenems are of particular interest since these drugs are frequently used in the treatment of Enterobacteriaceae infections. Extended-spectrum β-lactamases (ESBLs), in general, hydrolyze third-generation cephalosporins and aztreonam (a monobactam). The TEM, SHV, and CTX-M families are the most prominent ESBLs. Carbapenem-hydrolyzing β-lactamases (carbapenemases) hydrolyze the substrates of ESBLs and the carbapenems; ertapenem, imipenem, and meropenem. Two types of carbapenemases can be described: serine carbapenemases, which possess a serine moiety and can be inhibited by β-lactamase inhibitors (ie, clavulanate and tazobactam) and metallo-β-lactamases (MBLs), which require divalent cations, usually zinc, for enzyme activity and thus can be inhibited by EDTA and similar divalent cation chelators. The most prominent serine carbapenemases are those of the KPC (group A), Amp-C (group C), and OXA (group D) families and the most prominent MBLs belong to the IMP, VIM, and SPM families (group B). Study Design and Methods Using a combination of susceptibility testing, PCR targeting, and DNA sequencing, we characterized the phenotype and genotype of a carbapenem-resistant strain of K. pneumoniae (KpM) recovered from a pediatric patient in Seattle, Washington. Results KPC and TEM and the integron-related structures Int1, Int3, and Sul3 were amplified from KpM. Transformation of laboratory strains of Escherichia coli with KpM plasmid DNA yielded two daughter strains: KpM(KPC), which displays the carbapenemase phenotype, and KpM(TEM), which demonstrates the ESBL phenotype. KPC-2 and a TEM-type gene were amplified from KpM(KPC) and a TEM-type gene and Int3 were amplified from KpM(TEM). Conclusion These results (1) classified the genes responsible for the resistance pattern of KpM and (2) demonstrated that the resistance determinants were plasmid- and possibly integron-borne.

Conjugative Transfer of Multiresistance Plasmids from ESBL−positive Escherichia coli and Klebsiella spp. Clinical Isolates to Escherichia coli Strain K12 C600 Transfer koniugacyjny plazmidów wielooporności z ESBL−dodatnich klinicznych szczepów Escherichia coli i Klebsiella spp. do szczepu Escherichia coli K12 C600

Abstract: Objectives. The aim of this study was to evaluate the transfer frequency of plasmid−borne genes coding for extend− ed−spectrum β−lactamases (ESBLs) from clinical isolates of E. coli and Klebsiella spp. to the E. coli K12 C600 recipient strain. Additionally, the antimicrobial susceptibility of the donor strains and transconjugants obtained in mating experiments were studied. Material and Methods. A total of 51 ESBL−producing E. coli (n = 32) and Klebsiella spp. (n = 19) clinical strains isolated from children hospitalized in the Medical University Hospital in Wroclaw, Poland, were used in this study. Transfer of plasmids carrying ESBL−encoding genes was performed using the conjugational broth method. ESBL production was detected by the double−disk synergy test (DDST). The minimal inhibitory concentrations (MICs) of antimicrobial drugs were determined by an agar dilution technique on Mueller−Hinton agar. The presence of the blaCTX−M gene in donor strains and transconjugants was determined by PCR. Results. The majority of the isolates studied (92.2%) transferred ESBL−encoding plasmids to the E. coli K12 C600 recipient strain with a frequency of 10 −5 –10 –1 per donor strain. Donor strains and transconjugants displayed resis− tance patterns typical of ESBL producers. They were resistant to cefotaxime, cefrtiaxone, and aztreonam but sus− ceptible to carbapenems and oxyimino−β−lactams (ceftazidime, cefotaxime, ceftriaxone, and aztreonam) in combi− nation with clavulanic acid. Moreover, the majority of the strains exhibited a high level of resistance to non−β−lac− tam antimicrobials (gentamicin, amikacin, co−trimoxazole). The MIC values of cefotaxime and cefrtiaxone were significantly higher than those of ceftazidime, suggesting that this resistance may result from CTM−X−type ESBLs. PCR based on primers specific for CTX−M−type β−lactamases confirmed the presence of the blaCTX−M gene in 31 (66%) donor strains and 23 (48.9%) transconjugants. Conclusions. The majority of the strains tested harbored conjugative plasmids coding for CTX−M−type ESBLs. Additionally, genes conferring resistance to antimicrobial agents other than β−lactams were often co−transferred to the recipient strain in the conjugation process, indicating that these determinants were carried by ESBL−encoding plasmids (Adv Clin Exp Med 2007, 16, 2, 239–247).