Β-lactam Resistance Phenotypes Research Articles

Interaction of Ceftobiprole with the Low-Affinity PBP 5 of Enterococcus faecium

Ceftobiprole is a new cephalosporin that exhibits a high level of affinity for methicillin-resistant Staphylococcus aureus PBP 2a. It was reported that ceftobiprole did not interact with a mutated form of the low-affinity protein Enterococcus faecium PBP 5 (PBP 5fm) that, when overexpressed, confers a beta-lactam resistance phenotype to the bacterium. Our results show that ceftobiprole binds to unmutated PBP 5fm to form a stable acyl-enzyme and that ceftobiprole is able to efficiently kill a penicillin-resistant Enterococcus faecium strain that produces this protein.

Molecular typing and characterization of extended-spectrum TEM, SHV and CTX-M β-lactamases in clinical isolates of Enterobacter cloacae

Sixty-one non-repetitive Enterobacter cloacae ESBL producers were collected at the Amiens University Hospital in France. Eight β-lactam resistance phenotypes (a–h) and three aminoglycoside resistance phenotypes (i–k) were identified among these isolates, and 32 different pulsotypes were observed. Of these 61 isolates, 37 were sequenced and found to harbor β-lactamases with a pI of 5.9 (TEM-4), 6.5 (TEM-24), 7.8 (SHV-4), 8.2 (SHV-12), 8.4 (CTX-M-1) and 8.0 (CTX-M-9). Four imipenem-resistant ESBL-producing E. cloacae isolates did not express the 38kDa OMP, indicating that this resistance is associated with porin deficiency.

Detection and reporting β-lactam resistance phenotypes in Escherichia coli and Klebsiella pneumoniae: a multicenter proficiency study in Spain

The ability of 57 Spanish microbiology laboratories in detecting and reporting β-lactam resistance phenotypes in Escherichia coli and Klebsiella pneumoniae was evaluated. Laboratories received 6 well-characterized isolates expressing the most widespread extended-spectrum β-lactamases (ESBLs) in Spain (4 CTX-M type, 1 TEM type, and 1 SHV type), 3 isolates producing AmpC-type enzymes (2 plasmid mediated and 1 E. coli hyperproducing its chromosomal AmpC), and 3 quality control strains. Ninety-one percent of laboratories recognized all ESBL producers correctly, and therefore, low error rates were observed when testing cephalosporins and aztreonam. The highest error rates were observed with combinations of penicillin plus β-lactamase inhibitor, although more than 60% of cases were due to the interpretation made by the microbiologists. Correct recognition of all AmpC β-lactamase–producing strains occurred in only 47.4% of laboratories. These isolates were wrongly reported as ESBL producers and penicillinase hyperproducers in 7.6 % and 5.8% of cases, respectively. Detection of the AmpC-type phenotype by Spanish laboratories needs to be improved.

Impact of mecA promoter mutations on mecA expression and β-lactam resistance levels

The reason for the extremely low-level oxacillin resistance in a so-called ‘drug clone’, a methicillin-resistant Staphylococcus aureus circulating among injection drug users in Zurich, Switzerland, could be traced back to the mecA promoter sequence and particularly to the strain's genetic background. Sequencing of its mec complex identified a point mutation (TATA CT to TATA TT), creating a perfect palindrome in the −10 region of the mecA promoter/operator region containing the binding sites for the mecA repressors MecI and BlaI. Two strains with vastly different β-lactam resistance phenotypes, the low-level resistant drug clone type strain CHE482 and the highly homogeneously resistant strain COLn, were cured of their SCC mec elements and subsequently transformed with plasmids containing mecA under the control of either the wild-type or mutant promoter. Expression studies showed that this mutation had significant effects on both mecA transcription and corresponding PBP2a production, but only small effects on β-lactam resistance levels within a given genetic background. A further mutation in the mecA ribosomal binding site (GGAG G to GGAG T), common to SCC mec type IV strains, was found to have no discernable effect on mecA transcription and PBP2a content, and only minimal effects on β-lactam resistance. Factors associated with the genetic backgrounds into which these differently controlled mecA genes were introduced had a much higher impact on β-lactam resistance levels than the rates of mecA transcription. The tight repression of mecA expression in this drug clone in the absence of β-lactams could contribute to the apparent fitness of this fast growing strain.

Diversity of Extended-Spectrum β-Lactamases and Class C β-Lactamases among Cloacal Escherichia coli Isolates in Belgian Broiler Farms

A total of 295 ceftiofur-resistant Escherichia coli isolates were obtained from 489 cloacal samples collected at five different Belgian broiler farms with the aim to evaluate the diversity of this resistance at the farm level. Strains were examined for resistance against beta-lactam antibiotics and other antimicrobial agents by using disk diffusion tests. Three different beta-lactam resistance phenotypes suggested the presence of an extended-spectrum beta-lactamase (ESBL), a class C beta-lactamase, or the combination of an ESBL with a class C beta-lactamase. Seventy-six percent of these isolates also showed acquired resistance to other antimicrobial agents. After genotyping by repetitive extragenic palindromic-PCR, 51 unrelated E. coli strains were selected for further analyses. Isoelectric focusing and sequencing of the amplicons obtained in PCRs for the detection of genes encoding broad-spectrum beta-lactamase enzymes revealed the following ESBLs: TEM-52 (13.2%), TEM-106 (2%), CTX-M-1 (27.4%), CTX-M-2 (7.8%), CTX-M-14 (5.9%), and CTX-M-15 (2%). The only plasmidic AmpC beta-lactamase found in this study was the CMY-2 enzyme (49%). Mutations in the promoter and attenuator regions of the chromosomal ampC gene were found only in association with bla CMY-2 genes and ESBL genes. The combination of an ESBL (CTX-M-1) with a plasmidic AmpC beta-lactamase (CMY-2) was found in 7.8% of the isolates. These data show that ceftiofur-resistant E. coli strains are often present in cloacal samples of broilers at the farm level in Belgium. The diversity of broad-spectrum beta-lactamases among these isolates is high, and they may act as a reservoir of ESBL and ampC genes.

Influence of the β-lactam resistance phenotype on the cefuroxime versus cefditoren susceptibility of Streptococcus pneumoniae and Haemophilus influenzae recovered from children with acute otitis media

To study the influence of resistance phenotypes (based on sentinel antibiotics: penicillin and amoxicillin with/without clavulanate) on the cefuroxime versus cefditoren susceptibility of Streptococcus pneumoniae and Haemophilus influenzae recovered from children with acute otitis media. Middle ear isolates (193 S. pneumoniae and 114 H. influenzae) received in the Spanish Reference Laboratory (Instituto de Salud Carlos III) were tested. Antimicrobial susceptibility to penicillin, amoxicillin with/without clavulanate, cefuroxime and cefditoren was determined by agar dilution using Mueller-Hinton agar supplemented with 5% sheep blood for S. pneumoniae and Haemophilus Test Medium for H. influenzae. Strains were classified according to penicillin susceptibility (S. pneumoniae) or beta-lactamase production (H. influenzae). The decrease in penicillin susceptibility of S. pneumoniae (from the susceptible to the resistant category) decreased amoxicillin and cefuroxime susceptibility rates from 100% to 34% and 0%, respectively. All pneumococcal strains were inhibited by 0.5 mg/L cefditoren, including those from penicillin-resistant serotypes 14, 23F, 6B and 9V with higher amoxicillin versus penicillin MICs. Susceptibility rates of beta-lactamase-positive H. influenzae strains were 93.8% and 85.4% to amoxicillin/clavulanate and cefuroxime, respectively. Resistance to amoxicillin/clavulanate (MIC>or=8/4 mg/L) was 12.1% (8 out of 66) and 6.3% (3 out of 48) in beta-lactamase-negative and -positive strains, respectively. All H. influenzae strains were inhibited by <or=0.06 mg/L cefditoren. Susceptibility to sentinel beta-lactams cannot predict activity of other members of the group. The addition of clavulanic acid to amoxicillin does not guarantee 100% coverage of H. influenzae, regardless of beta-lactamase production.

Dissemination and persistence of a plasmid-mediated TEM-3-like β-lactamase, TEM-139, among Enterobacteriaceae in Bulgaria

During a survey of extended-spectrum β-lactamases (ESBLs) in Bulgaria from 1996 to 2003, a TEM-3-like ESBL was detected in strains of Klebsiella pneumoniae, Escherichia coli, Citrobacter freundii and Klebsiella oxytoca from three centres in three different towns. The nucleotide sequence of the cloned gene was identical to that of TEM-3, except for one substitution (C347A) causing an amino acid exchange at position 49 from leucine to methionine. This TEM-3 variant with both a unique nucleotide and amino acid sequence was designated TEM-139. Transformants producing TEM-3 or TEM-139 expressed identical β-lactam resistance phenotypes. TEM-139 was the only TEM-type ESBL detected in the surveyed hospitals (seven centres in three towns). TEM-139 is a natural variant of TEM-3 with an amino acid exchange without informational content, detectable only by molecular procedures, e.g. a nucleotide-specific polymerase chain reaction.

In Vivo Reversion to the Wild-Type β-Lactam Resistance Phenotype Mediated by a Plasmid CarryingampRandqnrA1inEnterobacter cloacae

Resistance to beta-lactams and quinolones in two isogenic Enterobacter cloacae isolates was studied. One was susceptible to cefoxitin and amoxicillin-clavulanate. The other one showed its natural beta-lactam resistance pattern. Both isolates had a nonfunctional AmpR regulator. However, within the second one, the presence of a plasmid carrying ampR and qnrA1 allowed reversion to the wild-type beta-lactam resistance phenotype and decreased susceptibility to fluoroquinolones.

Performance in detection and reporting β-lactam resistance phenotypes in Enterobacteriaceae: a nationwide proficiency study in Italian laboratories

We evaluated the ability of 60 Italian clinical microbiology laboratories in detecting and reporting β-lactam resistance phenotypes in Enterobacteriaceae. Laboratories received 5 well-characterized isolates producing extended-spectrum β-lactamases (ESBLs), 2 hyperproducers of chromosomal enzymes, and 3 quality control strains. The performances in antimicrobial susceptibility testing (AST) were different depending on the species and type of ESBL produced. High rates of very major errors (up to 56%) were observed for ESBL producers when testing cephalosporins and aztreonam, especially in the case of CTX-M-1–producing Escherichia coli and TEM-52–producing Proteus mirabilis. Isolates hyperproducing chromosomal enzymes were erroneously reported as ESBL producers in approximately 20% of cases. Detection of ESBLs is still a problem for clinical microbiology laboratories. Overall, performances in AST appear to be better with Klebsiella spp. producing well-known enzymes (e.g., SHV type) than with strains producing emerging enzymes (e.g., CTX-M type) or organisms not well recognized as ESBL producers (e.g., P. mirabilis).

β-Lactam resistance and β-lactamase expression in clinical Stenotrophomonas maltophilia isolates having defined phylogenetic relationships

To test the hypothesis that Stenotrophomonas maltophilia isolates from certain phylogenetic groups have predictable beta-lactamase expression and beta-lactam resistance profiles. Isolates were grouped using sequences of the 16S rRNA gene and smeT-smeD intergenic region. beta-Lactamase activities in cell extracts were quantified spectrophotometrically and beta-lactam MICs were determined using agar dilution methodology and Etest as appropriate. A collection of 50 clinical S. maltophilia isolates from Europe and North, South and Central America were phylogenetically grouped. Group 'A' (22 out of 50) includes remarkably genetically homogeneous isolates; group 'B' (17 out of 50) includes isolates that are genetically heterogeneous and quite distinct from those of group A. Members of these two groups are, however, indistinguishable in terms of their beta-lactam resistance and beta-lactamase expression phenotypes. In contrast, isolates from group 'C', which are less common (8 out of 50), are considerably more susceptible to beta-lactams owing to reduced inducibility of beta-lactamase expression following beta-lactam challenge. The majority of S. maltophilia clinical isolates behave similarly in terms of beta-lactamase expression and beta-lactam resistance properties, despite considerable phylogenetic variability.

TEM-109 (CMT-5), a Natural Complex Mutant of TEM-1 β-Lactamase Combining the Amino Acid Substitutions of TEM-6 and TEM-33 (IRT-5)

Escherichia coli CF349 exhibited a complex beta-lactam resistance phenotype, including resistance to amoxicillin and ticarcillin alone and in combination with clavulanate and to some extended-spectrum cephalosporins. The double-disk synergy test was positive. CF349 harbored an 85-kb conjugative plasmid which encoded a beta-lactamase of pI 5.9. The corresponding bla gene was identified by PCR and sequencing as a bla(TEM) gene. The deduced protein sequence revealed a new complex mutant of TEM-1 beta-lactamase designated TEM-109 (CMT-5). TEM-109 contained both the substitutions Glu104Lys and Arg164His of the expanded-spectrum beta-lactamase (ESBL) TEM-6 and Met69Leu of the inhibitor-resistant TEM-33 (IRT-5). TEM-109 exhibited hydrolytic activity against ceftazidime similar to that of TEM-6 (k(cat), 56 s(-1) and 105 s(-1), respectively; K(m) values, 226 and 247 microM, respectively). The 50% inhibitory concentrations of clavulanate and tazobactam (0.13 microM and 0.27 microM, respectively) were 5- to 10-fold higher for TEM-109 than for TEM-6 (0.01 and 0.06 microM, respectively) but were almost 10-fold lower than those for TEM-33. The characterization of this novel CMT, which exhibits a low level of resistance to inhibitors, highlights the emergence of this new ESBL type.

Genetic and biochemical characterization of a chromosome-encoded carbapenem-hydrolyzing ambler class D beta-lactamase from Shewanella algae.

A chromosome-encoded beta-lactamase gene from Shewanella algae clinical isolate KB-1 was cloned and expressed in Escherichia coli. It encoded the Ambler class D enzyme OXA-55, sharing less than 55% identity with any other oxacillinases. Although conferring a narrow-spectrum beta-lactam resistance phenotype, OXA-55 had carbapenem-hydrolyzing activity that mirrored the reduced susceptibility to imipenem observed in S. algae KB-1. Very similar oxacillinases were found in other S. algae isolates.

Antimicrobial resistance of Gram-negative bacilli isolates from inpatients and outpatients at Yaounde Central Hospital, Cameroon

Objective: To determine and compare antimicrobial susceptibility patterns of pathogenic bacteria from inpatients and outpatients at a university teaching hospital in Yaounde, Cameroon. Methods: Gram-negative bacilli isolates ( n=522), obtained from a wide range of clinical specimens (urine, pus and blood) from inpatients and outpatients at Yaounde Central Hospital between March 1995 and April 1998, were evaluated for resistance to antibiotics (amoxicillin, amoxicillin/clavulanate, piperacillin, cefazolin, cefoxitin, cefotaxime, ceftazidime, aztreonam, imipenem, gentamicin, tobramicin, ofloxacin and trimethoprim/sulfamethoxazole). Results: Of the 522 isolates recorded, 80.3% were Enterobacteriaceae. A high incidence of resistance to amoxicillin (85%), piperacillin (75%) and trimethoprim/sulfamethoxazole (71%) was observed. The proportion of antimicrobial-resistant isolates from inpatients was significantly higher than that from outpatients (P<0.05), except for piperacillin, tobramicin and trimethoprim/sulfamethoxazole. The combinations of antimicrobial and organism showed that the percentage of ceftazidime-resistant Pseudomonas aeruginosa and ceftazidime-resistant Enterobacter cloacae were 26.8% and 24% respectively. The rate of antimicrobial resistance in isolates from inpatients was not significantly higher than that in isolates from outpatients for all the antimicrobial/organism combinations, except for ceftazidime-resistant Escherichia coli, which was exclusively found in isolates from inpatients. Among Enterobacteriaceae, high and low level penicillinase (mostly in E. coli (13.6% and 11% respectively) and Klebsiella spp. (9% and 8% respectively) were the most important β-lactam resistance phenotypes (31.2% and 23.6%, respectively). Wild type (exclusively observed in E. coli, Proteus mirabilis and Salmonella spp.) and low level penicillinase were higher in outpatient than inpatient isolates (wild type—17.9% vs 10.8% and low level penicillinase—29.4% vs 20.5%, respectively; P<0.05). However, extended spectrum β-lactamase strains ( Klebsiella spp. (3.5%), E. coli (2.6%), Citrobacter spp. (0.7%), Enterobacter spp. (0.4%) and P. mirabilis (0.2%)) were exclusively recovered from inpatients. Penicillinase and high level cephalosporinase resistance phenotypes were frequently observed in non-fermenter Gram-negative bacilli (46.6% and 29.1% respectively). However, there were no significant differences in penicillinase and cephalosporinase resistance between inpatient and outpatient isolates. Conclusion: As the incidence of antimicrobial resistance is substantially higher in isolates from inpatient than outpatient pathogens, more resources should be allocated within the hospital to encourage good antibiotic practices and good hospital hygiene.

In vivo selection of a chromosomally encoded beta-lactamase variant conferring ceftazidime resistance in Klebsiella oxytoca.

Klebsiella oxytoca clinical isolate A was recovered from the urine of a 55-year-old man with prostatic and urinary tract infections. This isolate displayed a beta-lactam resistance phenotype consistent with overproduction of a chromosomally encoded class A beta-lactamase and had decreased susceptibilities to all beta-lactams except ceftazidime, cephamycins, and carbapenems. Four weeks after treatment with an antibiotic regimen that included ceftazidime, K. oxytoca isolate B, which had a high level of resistance to ceftazidime, was isolated from the urine of the same patient. Isoelectric focusing analysis of the culture extracts of these isolates gave a pI of 5.4 for both isolates. Cloning experiments with the PCR products of the bla(OXY) gene resulted in two Escherichia coli DH10B recombinant clones with resistance phenotypes mirroring those of the parental isolates. Sequencing analysis revealed that the bla(OXY-2-5) gene from K. oxytoca B had a single nucleotide substitution compared to the sequence of the bla(OXY-2) gene from K. oxytoca A, leading to a proline-to-serine substitution at position 167, according to the numbering of Ambler. Biochemical analysis of purified OXY-2-5 showed that it had the ability to hydrolyze ceftazidime. This is the first report of in vivo selection of a K. oxytoca isolate that produced a chromosomally encoded beta-lactamase conferring resistance to ceftazidime.

Emergence of Klebsiella pneumoniae isolates producing inducible DHA-1 beta-lactamase in a university hospital in Taiwan.

Ten nonrepetitive clinical isolates of Klebsiella pneumoniae exhibiting an unusual inducible beta-lactam resistance phenotype were identified between January 1999 and September 2001 in a university hospital in Taiwan. In the presence of 2 micro g of clavulanic acid, the isolates showed a one to four twofold concentration increase in the MICs of ceftazidime, cefotaxime, and aztreonam but remained susceptible to cefepime (MICs, </=0.5 micro g/ml) and imipenem (MICs, </=0.5 micro g/ml). PCR, sequence analysis, and isoelectric focusing revealed production by these isolates of TEM-1, SHV-11, and DHA-1, a plasmid-encoded inducible AmpC beta-lactamase originally found in a Salmonella enterica serovar Enteritidis strain. Transfer of the resistance by conjugation experiments was not successful, but Southern hybridization showed that bla(DHA-1) was located on 70-kb plasmids, suggesting that the bla(DHA-1)-containing plasmids in the K. pneumoniae isolates were non-self-transmissible. Five isolates were recovered from patients in two surgery wards and two intensive care units. Acquisition of the DHA-1 producers could be traced back to previous hospitalizations 1 to 5 months earlier for the other five patients. Six and seven patterns among the isolates were demonstrated by plasmid analysis and ribotyping, respectively, indicating that the spread of the DHA-1 producers was due to both horizontal transfer of bla(DHA-1) and dissemination of endemic clones.

Biological cost of AmpC production for Salmonella enterica serotype Typhimurium.

Chromosomally mediated AmpC-type beta-lactamases are frequently found among Enterobacteriaceae. Hyperproduction of AmpC beta-lactamase results in high-level resistance to beta-lactam antibiotics. One striking feature of Salmonella is the absence of the structural ampC gene, encoding AmpC beta-lactamase, in contrast with other members in the Enterobacteriaceae family, such as Escherichia, Citrobacter, or Enterobacter. The horizontal acquisition of ampC genes is one of the causes of the increased resistance to extended-spectrum cephalosporins and beta-lactamase inhibitors among gram-negative rods. Nevertheless, despite the high number of beta-lactam-resistant Salmonella isolates so far described, only two strains expressing resistance to cephalosporin and beta-lactamase inhibitors which is mediated by AmpC-type enzymes have been found. In this work, data are provided which support the possibility that the maintenance and expression of the ampC gene may represent an unbearable cost for Salmonella in terms of reduction of some of its lifestyle attributes, such as growth rate and invasiveness. The deleterious AmpC burden can be eliminated by decreasing the production of AmpC when both the regulatory gene, ampR, and ampC are present in Salmonella. Thus, it is suggested that the two genes have to be acquired together by Salmonella, leading to an inducible beta-lactam resistance phenotype. AmpC synthesis did not produce major variations in the peptidoglycan composition of Salmonella.

Evaluation of an expert system linked to a rapid antibiotic susceptibility testing system for the detection of β-lactam resistance phenotypes

Interpretive reading of antibiotic disc agar diffusion tests indicates the resistance mechanisms, if any, expressed by a bacterium. An expert system for determining resistance mechanisms using rapid automated antibiotic susceptibility tests has been developed. The β-lactan susceptibility of each of 300 strains of clinically significant species of enterobacteria, displaying natural and acquired resistance mechanisms, was determined by disc agar diffusion and by a rapid automated method of susceptibility testing associated with an expert system. For every strain, the conclusion of the expert analysis of the automated test was compared with the commonly accepted interpretation of disc agar diffusion tests.Of the 300 strains studied, 275 were similarly interpreted (91.7% agreement). The susceptible and naturally β-lactan-resistant phenotypes (wild phenotypes) were equally recognized by both methods. Similarly, the results of the two methods concurred for most of the acquired resistance phenotypes. However, for 25 strains (8.3%) the results diverged. The expert system proposed an erroneous phenotype (5 strains), several phenotypes including the correct one (17 strains), or no phenotype (1 strain). For 2 strains the natural resistance mechanism was not detected at first by the automated method but was subsequently deduced by the expert analysis according to bacterial identification.These results demonstrate that satisfactory interpretive reading of automated antibiotic susceptibility tests is possible in 4 to 5 hours but requires careful selection of the antibiotics tested as phenotypic markers.