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PubMed1997

A reassessment of the in-vitro activity of colistin sulphomethate sodium.

The in-vitro activity of colistin sulphomethate sodium was compared with that of other commonly used antimicrobial agents against 377 recent clinical isolates of Gram-negative bacteria (including 94 strains of Pseudomonas aeruginosa from patients with cystic fibrosis) and 16 organisms with defined resistance patterns. Colistin was active against most strains of P. aeruginosa (MIC90 4 mg/L), Shigella spp. (MIC90 0.5 mg/L), Salmonella spp. (MIC90 1 mg/L), Acinetobacter spp. (MIC90 2 mg/L), Citrobacter spp. (MIC90 1 mg/L), Escherichia coli (MIC90 1 mg/L), Klebsiella spp. (MIC90 8 mg/L) and Enterobacter spp. (MIC50 1 mg/L). No useful activity was demonstrated against Providentia spp. or Serratia spp. The results show that colistin remains a useful antimicrobial agent against Gram-negative bacteria, particularly those strains which are resistant to more commonly used antibiotics.

Anti-Bacterial Agents↗

PubMed1997

In-vitro activity of trovafloxacin against sensitive and resistant aerobic bacteria using the standard microdilution broth method and Etest.

A comparison of MICs of trovafloxacin (CP-99,219) determined by the standard microdilution broth method versus the Etest was performed for multiple strains of Gram-positive and Gram-negative bacteria. A comparison was also made of the in-vitro activity of trovafloxacin versus ciprofloxacin and ofloxacin. The MIC50 and MIC90 were determined by both methods for each species tested. The Etest resulted in MICs one to two dilutions higher than the microdilution broth method. Trovafloxacin was the most active agent against Gram-positive organisms. Ciprofloxacin was the most active agent against Citrobacter freundii, Proteus mirabilis, Proteus vulgaris, Morganella morganii and Serratia marcescens, while trovafloxacin had equal or greater activity compared with ciprofloxacin and ofloxacin against the other Gram-negative organisms tested. Overall, ofloxacin was the least active agent tested. In addition, the in-vitro activity of trovafloxacin or ciprofloxacin in combination with ampicillin/sulbactam, gentamicin or vancomycin was evaluated. The combination of trovafloxacin and gentamicin was synergic against two of 20 Enterococcus faecium isolates, the combination of trovafloxacin and ampicillin/sulbactam was synergic against two of 24 Enterococcus faecalis isolates, and the combination of ciprofloxacin and gentamicin was synergic against one of 25 Stenotrophomonas maltophilia isolates. All other antibiotic combinations resulted in an additive or indifferent effect.

Ampicillin↗

PubMed1998

Beta-lactamase-mediated resistance and opportunities for its control.

Clinical use of beta-lactams has selected for beta-lactamase-producing organisms. Numerous beta-lactamases are known, and sequencing allows them to be divided into four Classes, A to D, with Classes A and C being the most important. Pharmaceutical chemists have responded to the spread of beta-lactamase-producing organisms by developing stable agents and inhibitors. Stability in penicillins and cephalosporins is achieved by attaching a bulky substituent to the amino group of 6-aminopenicillanic acid or 7-aminocephalosporanic acid, or by replacing the hydrogen on carbon 6 (penicillins) or 7 (cephalosporins) with an alpha-methoxy group. In carbapenems, stability is achieved by incorporation of a simple trans-6-hydroxyethyl group. Beta-lactamase-inhibitory activity occurs in many beta-lactam classes but only clavams and penicillanic acid sulphones have been developed specifically as beta-lactamase inhibitors. These inhibit most Class A and some Class D enzymes but act poorly against Class B and C enzymes. Their success is affected by the amount of enzyme, the permeability of the bacterial cell wall, the partner beta-lactam and the pH. Piperacillin/tazobactam, which combines a good inhibitor of Class A enzymes with a broad-spectrum, easily-protected penicillin, has wide activity against common pathogens, the major exceptions being strains of Enterobacter, Serratia and Citrobacter freundii that produce large amounts of Class C enzymes, and Gram-positive cocci with modified penicillin-binding proteins. Beta-lactamase-stable beta-lactams and inhibitor combinations overcome many existing resistance mechanisms but are themselves selecting new resistances. Few new beta-lactams able to overcome these resistances are advanced in development and consequently the opportunities for control lie mostly in the more prudent use of compounds already available.

Anti-Bacterial Agents↗

PubMed1999

Concurrent outbreaks of extended-spectrum beta-lactamase-producing organisms of the family Enterobacteriaceae in a Warsaw hospital.

The increasing use of broader-spectrum cephalosporins in the first half of the 1990s has become one of the major factors responsible for the high rate of selection of extended-spectrum beta-lactamase (ESBL)-producing microorganisms in Polish hospitals. Thirty-five isolates of seven different species of the family Enterobacteriaceae were identified as ESBL producers, over a 4 month period, in one of Warsaw's hospitals between the end of 1996 and the beginning of 1997. Sixteen per cent of all Klebsiella pneumoniae isolates, 16% of Citrobacter freundii isolates and 32% of Serratia marcescens isolates collected by the hospital microbiology laboratory at that time were expressing these enzymes. The majority of these (27 isolates) were found to express CTX-M-type ESBLs (pI 8.4). This outbreak was due to both plasmid dissemination among unrelated strains and clonal spread of some strains in several wards of the hospital. The remaining isolates produced ESBLs (pI 8.2) belonging to the SHV family of beta-lactamases and demonstrated a high degree of genetic diversity.

Bacterial Typing Techniques↗

PubMed2000

In vitro activity of gemifloxacin against a broad range of recent clinical isolates from the USA.

The antibacterial potencies of gemifloxacin (SB-265805) and 13 comparator compounds were determined by broth microdilution against a panel of 645 Gram-positive and 995 Gram-negative organisms collected from various USA sites. Time-kill studies were performed and postantibiotic effect (PAE) was determined for several organisms using trovafloxacin and ciprofloxacin as comparator compounds. Based on MIC(90)s, gemifloxacin was the most potent compound tested against Gram-positive isolates: Streptococcus pneumoniae (MIC(90) 0. 016 mg/L), Streptococcus agalactiae (0.03 mg/L), Streptococcus pyogenes (0.03 mg/L), viridans streptococci (0.12 mg/L), methicillin-susceptible Staphylococcus aureus (0.03 mg/L), Staphylococcus epidermidis (2 mg/L), Staphylococcus saprophyticus (0. 016 mg/L) and Enterococcus faecalis (2 mg/L). Against Gram-negative isolates, the potency of gemifloxacin was equal to that of levofloxacin and ciprofloxacin and generally better than that of ofloxacin, grepafloxacin, trovafloxacin and nalidixic acid. MIC(90)s for gemifloxacin were: Haemophilus influenzae (</=0.008 mg/L), Moraxella catarrhalis (0.008 mg/L), Escherichia coli (0.016 mg/L), Klebsiella pneumoniae (0.25 mg/L), Klebsiella oxytoca (0.25 mg/L), Enterobacter cloacae (1 mg/L), Enterobacter aerogenes (0.25 mg/L), Proteus spp. (4 mg/L), Serratia spp. (1 mg/L), Citrobacter freundii (2 mg/L), Morganella morganii (0.12 mg/L), Pseudomonas aeruginosa (8 mg/L), Stenotrophomonas maltophilia (4 mg/L) and Acinetobacter spp. (32 mg/L). Gemifloxacin was bactericidal for all organisms studied at 2 and 4 x MIC. The PAE for most strains was in the range 0.7-2.5 h at 2 and 4 x MIC, although longer PAEs were observed with H. influenzae, P. aeruginosa and Proteus vulgaris (>6 h at 4 x MIC) and shorter PAEs with E. faecalis (0.1-0.6 h) and K. pneumoniae (0.1-0.2 h). In conclusion, gemifloxacin is a novel quinolone with a broad spectrum of antimicrobial activity. It has substantially improved potency against Gram-positive organisms, especially streptococci, for which gemifloxacin is generally at least eight- to 16-fold more potent than other quinolones tested. It retains the good Gram-negative activity seen with ciprofloxacin and levofloxacin and shows good bactericidal activity and prolonged PAEs.

Anti-Infective Agents↗

PubMed2001

SHV-12, SHV-5, SHV-2a and VEB-1 extended-spectrum beta-lactamases in Gram-negative bacteria isolated in a university hospital in Thailand.

Sixty-one extended-spectrum beta-lactamase (ESBL)-producing isolates were collected from Srinagarind Hospital, Thailand. These included 43 Enterobacteriaceae and 18 Pseudomonadaceae. The 43 Enterobacteriaceae were found to produce the following ESBLs: 26 (60.5%) SHV-12, 13 (30.2%) SHV-5, two (4.7%) SHV-2a, one (2.3%) VEB-1 and one (2.3%) unidentified. Twenty-four isolates (55.8%) also carried bla(TEM-1B), as well as bla(SHV) or bla(VEB-1). Plasmid DNA from transconjugants carrying the bla(SHV-12) gene showed various restriction patterns, indicating the distribution of the bla(SHV-12) gene among different antibiotic resistance plasmids. In contrast, bla(SHV-5) in 13 isolates was found on a single plasmid of c. 130 kb. Pulsed-field gel electrophoresis (PFGE) analysis of genomic DNA from these isolates revealed that nine of 11 Klebsiella pneumoniae gave the same pattern, indicating clonal spread of the strain within the hospital, together with the occasional spread of the plasmid to other strains. Among the pseudomonad isolates, 16 Pseudomonas aeruginosa and one Pseudomonas putida had bla(VEB-like) and one P. aeruginosa had bla(SHV-12). Nine of the 16 isolates carrying bla(VEB-like) (56.3%) had identical PFGE patterns, suggesting the dissemination of this gene, also by clonal spread. At least six different bla(VEB-like-)containing integrons were found among the 18 isolates. This is the first report of bacteria producing SHV-12 and SHV-2a in Thailand and the first report of SHV-12 in P. aeruginosa, of VEB-1 in Citrobacter freundii and a VEB-1-like beta-lactamase in P. putida. These findings indicate that ESBL genes in the Far East are part of a gene pool capable of broad horizontal gene transfer, in that these genes can transfer between different families of Gram-negative bacilli.

Cross Infection↗

PubMed2001

Detection of beta-lactamase-mediated resistance.

beta-Lactams are the most widely used antibiotics, and beta-lactamases are the greatest source of resistance to them. An understanding of beta-lactamase detection and identification is therefore valuable. Colorimetric, acidimetric and iodometric tests of beta-lactamase production are good, rapid indicators of penicillin and ampicillin resistance in Haemophilus, Moraxella and Neisseria spp. These methods can also be applied to Gram-negative aerobic bacilli but are less useful, since the usual question is not whether a beta-lactamase is produced by these organisms, but which beta-lactamase? Accurate identification of the beta-lactamases of Enterobacteriaceae demands gene or protein sequencing, but the broad type of enzyme produced by an isolate can often be inferred from antibiotic susceptibility data. Resistance to ceftazidime or cefpodoxime implies extended-spectrum beta-lactamase (ESBL) production in Escherichia coli and Klebsiella spp., especially if susceptibility to cefoxitin is retained. ESBL production can be confirmed with double disc tests or with various commercial kits. Derepression of AmpC beta-lactamases in Enterobacter spp. and Citrobacter freundii is another important mechanism and can be inferred from cross-resistance to beta-lactamase inhibitor combinations and to all cephalosporins except fourth-generation agents. Antagonism between cefoxitin and cefotaxime can be used to infer the presence of inducible AmpC enzymes in these species, indicating the risk of segregation of derepressed mutants, but in general this risk is better predicted from accurate speciation.

Bacteria↗

PubMed2002

Antimicrobial susceptibilities among clinical isolates of extended-spectrum cephalosporin-resistant Gram-negative bacteria in a Taiwanese University Hospital.

Infections caused by Gram-negative bacteria with resistance to extended-spectrum cephalosporins require the identification of effective alternative antimicrobial therapy. To determine the role of other pre-existing or currently available antimicrobial agents in treating infections caused by these multidrug-resistant pathogens, we evaluated the in vitro susceptibilities of these agents in 411 non-duplicate isolates of extended-spectrum cephalosporin-resistant Gram-negative bacteria recovered between January 1999 and December 1999 in a major teaching hospital in Taipei, Taiwan. These isolates included cefotaxime-resistant (MICs > or = 2 mg/L) Escherichia coli (66 isolates) and Klebsiella pneumoniae (77 isolates); cefotaxime-resistant (MICs > or = 64 mg/L) Enterobacter cloacae (59 isolates), Serratia marcescens (52 isolates) and Citrobacter freundii (52 isolates); and ceftazidime-resistant (MICs > or = 64 mg/L) Pseudomonas aeruginosa (50 isolates) and Acinetobacter baumannii (55 isolates). Overall, carbapenems (imipenem and meropenem) had good activity against the cefotaxime-resistant Enterobacteriaceae tested (>90% of isolates were susceptible). However, carbapenems had limited activity against the ceftazidime-resistant P. aeruginosa (only 4% of isolates were susceptible) and A. baumannii (51-56% of isolates were susceptible). Among the E. coli and K. pneumoniae isolates tested, 33.3% and 58.4%, respectively, exhibited extended-spectrum beta-lactamase phenotype, determined by the double disc method. Over 80% of cefotaxime-resistant E. cloacae and C. freundii were susceptible to cefepime, but this agent had limited activity against other bacteria tested. Susceptibilities of these isolates to ciprofloxacin varied, ranging from 25% for A. baumannii to 92% for E. cloacae. Newer fluoroquinolones (moxifloxacin and trovafloxacin) had equal or less activity against these organisms, except for A. baumannii for which their MIC(90)s (8-16 mg/L) were four- to 16-fold less than that of ciprofloxacin (MIC(90) 128 mg/L).

Cefotaxime↗

PubMed2003

Analyses of ampC gene expression in Serratia marcescens reveal new regulatory properties.

Serratia marcescens encodes an inducible, chromosomal beta-lactamase, ampC. Studies addressing the regulation of inducible ampC genes have focused primarily on Enterobacter cloacae and Citrobacter freundii. The purpose of this study was to clone and sequence the ampC, ampR and intergenic region of S. marcescens and examine both inducible and basal level ampC expression. Sequence analysis of the S. marcescens ampC gene identified an extended 5' untranslated region (UTR) of 126 nucleotides, which formed a prominent stem-loop structure. Induction of ampC expression required AmpR, and the start of transcription was determined using primer extension analysis. In vivo half-life analysis revealed that the half-life of the S. marcescens ampC transcript was 7 min. Confirmation of the in vivo half-life and the role of the stem-loop structure in the 5' UTR was demonstrated by comparing transcript half-life and luciferase expression between a wild-type (WT) and a 5' UTR stem-loop deletion mutant. These data demonstrated that the stem-loop structure was involved in transcript stability. Taken together, these findings indicate that constitutive expression of S. marcescens ampC is regulated by both transcriptional initiation and post-transcriptional events.

5' Untranslated Regions↗

PubMed2004

Plasmid-encoded functions compensate for the biological cost of AmpC overexpression in a clinical isolate of Salmonella typhimurium.

OBJECTIVES: In a previous study with a Salmonella typhimurium strain containing cloned ampC-ampR from Enterobacter cloacae, it was suggested that ampC expression must be kept at low levels by AmpR to maintain normal growth and virulent phenotype. The purpose of this study was to determine whether findings obtained with a laboratory model can be extended to a virulent clinical isolate of S. typhimurium expressing the plasmid-encoded bla(CMY-7). METHODS: Disc induction assays were carried out to investigate inducibility of bla(CMY-7). Primer extension and sequence analyses were carried out to map the transcriptional start site of bla(CMY-7) and determine the relative expression. Growth and invasion potential of Salmonella strains were monitored by optical density, viable counts and cell invasion assays. RESULTS: Sequence analysis confirmed the absence of ampR upstream of bla(CMY-7) therefore confirming the negative results observed using the disc induction assay. Primer extension analysis mapped the start site of bla(CMY-7) transcription within an ISEcp1-like element. The relative expression of bla(CMY-7) was approximately 965-fold higher than the expression of a wild-type Citrobacter freundii chromosomal ampC and approximately 4.1-fold higher than ampC expression from a derepressed mutant of C. freundii. Growth and the capacity to invade mammalian cells were not compromised for either the clinical isolate or the S. typhimurium transconjugant containing bla(CMY-7). However, a Salmonella transformant containing bla(CMY-7) exhibited a compromised phenotype with respect to growth and invasion of mammalian cells. CONCLUSION: These findings indicate that the biological cost of high-level AmpC production can be compensated by plasmid-encoded factors and not by regulating ampC expression.

Cell Line, Tumor↗

PubMed2004

In vitro activity of AVE1330A, an innovative broad-spectrum non-beta-lactam beta-lactamase inhibitor.

OBJECTIVES: Production of beta-lactamases is the main mechanism of beta-lactam resistance in Gram-negative bacteria. Despite the current use of clavulanic acid, sulbactam and tazobactam, the prevalence of class A and class C enzymes is increasing worldwide, demanding new beta-lactamase inhibitors. Here we report the antimicrobial properties of AVE1330A, a representative of a novel class of bridged bicyclico[3.2.1]diazabicyclo-octanones in combination with ceftazidime. MATERIALS AND METHODS: IC(50) and kinetic parameters of the hydrolysis reaction were used to characterize beta-lactamase inhibition by AVE1330A. MICs for >600 strains were determined with the combination ceftazidime/AVE1330A at a fixed ratio of 4:1. RESULTS: IC(50)s of AVE1330A for TEM-1 and P99 enzymes were 0.0023 mg/L (8 nM) and 0.023 mg/L (80 nM), compared with 0.027 mg/L (130 nM) and 205.1 mg/L (1 x 10(6) nM) of clavulanic acid and 0.013 mg/L (40 nM) and 1.6 mg/L (5000 nM) of tazobactam. A highly stable covalent complex led to a low turnover of AVE1330A. MICs of ceftazidime/AVE1330A for Enterobacteriaceae were at least eight-fold lower than those of ceftazidime alone. All of the Escherichia coli, Klebsiella pneumoniae, Citrobacter and Proteus mirabilis strains, including ceftazidime-resistant isolates, were inhibited at 4-8 mg/L. Only 2 mg/L were required to inhibit other Proteeae, Enterobacter, Salmonella and Serratia. CONCLUSION: The combination of ceftazidime with AVE1330A exhibited broad-spectrum activity against Ambler class A- and class C-producing Enterobacteriaceae.

Azepines↗

PubMed2004

Frequency and diversity of Class A extended-spectrum beta-lactamases in hospitals of the Auvergne, France: a 2 year prospective study.

OBJECTIVES: To evaluate the frequency and diversity of extended-spectrum beta-lactamases (ESBLs) produced by Enterobacteriaceae and Pseudomonas aeruginosa in one French region. METHODS: During 2001-2002, all the non-duplicate isolates of P. aeruginosa resistant to ceftazidime and of Enterobacteriaceae intermediate or resistant to ceftazidime and/or cefotaxime and/or aminoglycosides with an AAC(6') I phenotype were collected in nine hospitals of the area. ESBL isoelectric points were determined, bla genes were amplified and sequenced and epidemic isolates were genotyped with ERIC2-PCR. RESULTS: ESBLs were observed in 297 Enterobacteriaceae (0.8%). The most frequent were TEM-3 like (n=152; 51.2%) and TEM-24 (n=115; 38.7%). Four new enzymes were observed, TEM-112 (pI 5.4), TEM-113 (pI 6.3), TEM-114 (pI 5.9) and TEM-126 (pI 5.4). Other TEMs were TEM-8, TEM-12, TEM-16, TEM-19, TEM-20, TEM-21, TEM-29 and TEM-71. The other ESBLs were SHV-4, SHV-5 and SHV-12, CTX-M-1, CTX-M-3, CTX-M-14 and CTX-M-15. In 37 P. aeruginosa (0.7%) only one ESBL was observed, PER-1. Five epidemic strains were detected, Serratia marcescens TEM-3 and four observed in several hospitals, Enterobacter aerogenes TEM-24, Citrobacter koseri TEM-3, Proteus mirabilis TEM-3 and P. aeruginosa PER-1. CONCLUSION: ESBL frequency was lower than in 1998, and CTX-M-type frequency higher (2.1% of ESBLs in 2001, 4.9% in 2002). This long-term survey detected new sporadic enzymes (TEM-112, TEM-113, TEM-114 and TEM-126) and interhospital epidemic strains while avoiding any overestimation of ESBL frequency that may otherwise have occurred because of acute epidemics.

Amino Acid Substitution↗

PubMed2004

Predominance and genetic diversity of community- and hospital-acquired CTX-M extended-spectrum beta-lactamases in York, UK.

OBJECTIVES: This study was conducted to detect the presence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae within the faecal flora of both community- and hospital-based patients in York and to characterize the bla(TEM), bla(SHV) and bla(CTX-M) genes present in these isolates. METHODS: One thousand faeces samples were collected and screened at York Hospital during October-December 2003. Ninety-five non-duplicate Enterobacteriaceae isolates resistant to third-generation cephalosporins were recovered; 22 isolates were selected for further study on the basis of a positive double disc diffusion test for ESBL production. Antibiotic susceptibility testing was performed to a range of antibiotics. The TEM, SHV and CTX-M genes were detected by PCR and the DNA sequenced. RESULTS: The distribution of ESBL-positive isolates from the hospital and community was 1.4:1. These included nine Escherichia coli, seven Enterobacter cloacae, four Citrobacter freundii and a single isolate each of Klebsiella spp. and Salmonella spp. A total of 17 isolates contained bla(CTX-M) (five bla(CTX-M-15), three bla(CTX-M-14) and nine bla(CTX-M-9)). ISEcp1 was present in isolates expressing CTX-M-14 and -15, but was absent upstream of In60-associated bla(CTX-M-9). E. coli isolates also contained either a bla(TEM-1) or bla(TEM-2), whereas six of the E. cloacae carried bla(SHV-12) and the Klebsiella spp. bla(SHV-36) in addition to bla(CTX-M-9). The single Salmonella spp. carried bla(SHV-12). CONCLUSIONS: The overall prevalence of ESBL in isolates of Enterobacteriaceae from York was 1.9%. ESBL-producing isolates were found in both the community and hospital, with the CTX-M type most common. This is also the first report of an ESBL-producing Salmonella in the UK.

Community-Acquired Infections↗

PubMed2004

Evaluation of the MicroScan ESBL plus confirmation panel for detection of extended-spectrum beta-lactamases in clinical isolates of oxyimino-cephalosporin-resistant Gram-negative bacteria.

OBJECTIVE: We aimed to assess the performance of the MicroScan ESBL plus confirmation panel using a series of 87 oxyimino-cephalosporin-resistant Gram-negative bacilli of various species. METHODS: Organisms tested included 57 extended-spectrum beta-lactamase (ESBL) strains comprising Enterobacter aerogenes (3), Enterobacter cloacae (10), Escherichia coli (11), Klebsiella pneumoniae (26), Klebsiella oxytoca (3) and Proteus mirabilis (4). Also included were 30 strains resistant to oxyimino cephalosporins but lacking ESBLs, which were characterized with other resistance mechanisms, such as inherent clavulanate susceptibility in Acinetobacter spp. (4), hyperproduction of AmpC enzyme in Citrobacter freundii (2), E. aerogenes (3), E. cloacae (3), E. coli (4), Hafnia alvei (1) and Morganella morganii (1), production of plasmid-mediated AmpC beta-lactamase in K. pneumoniae (3) and E. coli (3) or hyperproduction of K1 enzyme in K. oxytoca (6). RESULTS: The MicroScan MIC-based clavulanate synergy correctly classified 50 of 57 ESBL strains as ESBL-positive and 23 of 30 non-ESBL strains as ESBL-negative (yielding a sensitivity of 88% and a specificity of 76.7%, respectively). False negatives among ESBL producers were highest with Enterobacter spp. due to masking interactions between ESBL and AmpC beta-lactamases. False-positive classifications occurred in two Acinetobacter spp., one E. coli producing plasmid-mediated AmpC beta-lactamase and two K. oxytoca hyperproducing their chromosomal K1 beta-lactamase. CONCLUSION: The MicroScan clavulanate synergy test proved to be a valuable tool for ESBL confirmation. However, this test has limitations in detecting ESBLs in Enterobacter spp. and in discriminating ESBL-related resistance from the K1 enzyme and from inherent clavulanate susceptibility in Acinetobacter spp.

Cephalosporin Resistance↗

PubMed2005

Genetic basis for dissemination of armA.

OBJECTIVES AND METHODS: armA is a novel plasmid-borne 16S rRNA methyltransferase that confers high-level resistance to 4,6-disubstituted deoxystreptamines. Recently, we have isolated from a high-level broad-spectrum aminoglycoside-resistant Escherichia coli animal isolate a plasmid, pMUR050, that bore the armA gene. In order to elucidate the genetic basis for the spread of armA, we have determined the complete nucleotide sequence of pMUR050. RESULTS: armA was borne by a complex transposon composite flanked by two direct repeats of IS26. The transposon composite included a class one integron with sul1 for resistance to sulphonamides and ant3''9 conferring resistance to spectinomycin-streptomycin, and a macrolide efflux pump and mefE/mel conferring high-level resistance to erythromycin. We identified in GenBank that another plasmid, pCTX-M3, from a Polish Citrobacter freundii human isolate, bore the same genetic structure, including armA. CONCLUSIONS: armA is present in human and animal isolates within a novel transposon composite. Further spread of armA between bacteria of diverse origin is to be expected.

Aminoglycosides↗

PubMed2005

High prevalence of the plasmid-mediated quinolone resistance determinant qnrA in multidrug-resistant Enterobacteriaceae from blood cultures in Liverpool, UK.

OBJECTIVES: To determine the prevalence of the plasmid-mediated quinolone resistance qnrA gene in a selected collection of blood culture isolates of Enterobacteriaceae resistant to both ciprofloxacin and cefotaxime. METHODS: Over a 29 month period, a total of 47 non-repetitive isolates of Enterobacteriaceae resistant to both ciprofloxacin and cefotaxime were identified. Isolates were screened for the presence of the qnrA gene, class I integrons and bla(ESBL) by PCR. Transferability was examined by conjugation with the sodium azide-resistant Escherichia coli J53. All qnrA-positive isolates were examined for DNA-relatedness by PFGE. RESULTS: A total of 15 of the 47 test isolates (32%) were positive for the qnrA gene, and included single isolates of E. coli and Citrobacter freundii, 4 Klebsiella pneumoniae and 9 Enterobacter cloacae. All 15 qnrA-positive isolates carried class 1 integrons, and 11 the extended-spectrum beta-lactamase gene bla(SHV-12). By PFGE two K. pneumoniae and three E. cloacae, respectively, were considered clonally but not temporally related. Plasmid transfer of quinolone resistance was only achieved with single isolates of K. pneumoniae and E. cloacae. Both plasmids carried class 1 integrons with a pSAL-1-like gene cassette arrangement intl1-aadA2-qacEDelta-sul1. CONCLUSIONS: In this selected group of ciprofloxacin- and cefotaxime-resistant bacteria, carriage of the qnrA gene was high (32%). This compares with <2.0% as demonstrated in worldwide studies of laboratory collections of ciprofloxacin-resistant bacteria. The majority of qnrA-positive isolates in our study originated from high-dependency care units within our hospital, but were shown not to be clonal by PFGE. This is the first report of qnrA-positive Enterobacteriaceae in the United Kingdom.

Anti-Bacterial Agents↗

PubMed2005

Surveillance of extended-spectrum beta-lactamases from clinical samples and faecal carriers in Barcelona, Spain.

OBJECTIVES: The aim of the present study was to characterize and compare the extended-spectrum beta-lactamase (ESBL)-producing organisms isolated from clinical samples and faecal carriers in 2001 and 2002. METHODS: A total of 5251 Enterobacteriaceae isolated from clinical samples and 1321 stool samples were evaluated for the presence of ESBLs. The stool samples were spread onto plates of MacConkey agar containing 2 mg/L cefotaxime for selection of ESBL-producing strains. These strains were defined as those showing synergism between amoxicillin/clavulanic acid and third-generation cephalosporins. The beta-lactamases involved were characterized by isoelectric focusing, PCR assays and DNA sequencing. RESULTS: The prevalence of ESBL-producing strains among clinical Enterobacteriaceae was 1.7%. Of these, 87.6% produced CTX-M, 25.8% produced SHV and 2.2% were TEM-type-producing strains. All clinical ESBL-producing strains were Escherichia coli, with the exception of four Klebsiella pneumoniae and one Citrobacter freundii. The prevalence of faecal carriage of ESBL-producing organisms was 3.3%. Of these, 75% produced CTX-M-type enzymes followed by 22.7% SHV-producing strains. All faecal ESBL-producing strains were E. coli except for one Enterobacter cloacae and one Proteus mirabilis. This latter strain produced the PER-1 enzyme reported for the first time in Spain. CONCLUSIONS: The prevalence of ESBL-producing strains in stool samples was higher than that observed in clinical samples from the same period. The different types of ESBLs found were similar in both contexts. The most prevalent ESBLs were the CTX-M-related enzymes, with nine different types, followed by SHV-12.

Carrier State↗

PubMed2005

Effect of 1-(1-naphthylmethyl)-piperazine, a novel putative efflux pump inhibitor, on antimicrobial drug susceptibility in clinical isolates of Enterobacteriaceae other than Escherichia coli.

OBJECTIVES: 1-(1-Naphthylmethyl)-piperazine (NMP) has been shown to reverse multidrug resistance (MDR) in Escherichia coli overexpressing RND type efflux pumps, but there is no data on its activity in Enterobacteriaceae other than E. coli. METHODS: The antimicrobial susceptibilities of laboratory strains and 167 clinical isolates of Enterobacteriaceae to a variety of antimicrobial agents were determined in the absence and presence of NMP and, for comparison, of Phe-Arg-beta-naphthylamide (PAbetaN), another putative efflux pump inhibitor (EPI). A 4-fold or greater reduction of the MIC after EPI addition was considered significant. RESULTS: NMP consistently reduced the MIC of linezolid in Citrobacter freundii, Enterobacter aerogenes and Klebsiella pneumoniae clinical isolates. Significant effects of NMP addition in >50% of tested isolates were also seen for levofloxacin, tetracycline and chloramphenicol in E. aerogenes, and for levofloxacin and tetracycline in K. pneumoniae, whereas no or minor effects were observed in Serratia marcescens. MDR reversal by NMP was more likely in isolates with decreased susceptibility to fluoroquinolones. In most fluoroquinolone-resistant strains the activity was sufficient to render isolates drug-susceptible at clinically achievable concentrations. The activity of PAbetaN was different from that of NMP, suggesting different modes of action of the two putative EPIs. CONCLUSION: NMP has moderate activity in reversing MDR in many but not all members of the Enterobacteriaceae family including clinical isolates. Its effects on resistance reversal depend on bacterial species and drug, and are different from those seen with PAbetaN.

Anti-Bacterial Agents↗