In-vitro susceptibility of thirty strains of Haemophilus ducreyi to several antibiotics including six cephalosporins

We have studied the in-vitro susceptibility of 30 strains of Haemophilus ducreyi to 22 antibacterial agents. Twenty-seven strains produced a beta-lactamase that had an isoelectric point similar to the isoelectric point of the TEM 1 type beta-lactamase produced by pSF 2124. Except for cefamandole, the 30 strains had similar susceptibilities to the different cephalosporins, cefotaxime being the most active compound. Twenty-two isolates were resistant to tetracycline and doxycycline; minimal inhibitory concentration (MIC) of minocycline was 4 mg/l for all the strains. One isolate was resistant to chloramphenicol (MIC = 16 mg/l). All strains were relatively resistant to colistin and nalidixic acid, and susceptible to streptomycin, kanamycin, gentamicin, erythromycin, rifampicin and vancomycin.     

Mutants of the TEM-1 beta-lactamase conferring resistance to ceftazidime

Spontaneous ceftazidime resistant mutants were obtained from an Escherichia coli K12 J62-2 expressing the TEM-1 beta-lactamase (mutation frequency = 10(-9). These mutants produced beta-lactamases with similar molecular weights, kinetic parameters and isoelectric points (pI) to the beta-lactamases produced by ceftazidime resistant clinical isolates which have recently been identified in this laboratory. Mutant enzyme A focused as a doublet band at pI 5.3 with an additional weak pI 5.4 band. The doublet co-focused with the TEM-E2 beta-lactamase, produced by a ceftazidime resistant Klebsiella oxytoca isolate, which was originally obtained in a Liverpool hospital. Mutant enzyme B had a pI identical to the TEM-E1 beta-lactamase produced by a ceftazidime resistant clinical isolate of E. coli found in Belgium. These results suggest that the two beta-lactamases in the clinical strains may have come from simple mutations of the TEM-1 beta-lactamase gene.     

Pseudomonas aeruginosa isolates from patients with cystic fibrosis have different beta-lactamase expression phenotypes but are homogeneous in the ampC-ampR genetic region.

Pseudomonas aeruginosa isolates from 1 of 17 cystic fibrosis patients produced secondary beta-lactamase in addition to the ampC beta-lactamase. Isolates were grouped into three beta-lactamase expression phenotypes: (i) beta-lactam sensitive, low basal levels and inducible beta-lactamase production; (ii) beta-lactam resistant, moderate basal levels and hyperinducible beta-lactamase production; (iii) beta-lactam resistant, high basal levels and constitutive beta-lactamase production. Apart from a base substitution in the ampR-ampC intergenic region of an isolate with moderate-basal-level and hyperinducible beta-lactamase production, sensitive and resistant strains were identical in their ampC-ampR genetic regions. Thus, enhanced beta-lactamase expression is due to mutations in regulatory proteins other than AmpR.     

Activity of cefepime against ceftazidime- and cefotaxime-resistant gram-negative bacteria and its relationship to beta-lactamase levels.

One hundred clinical isolates resistant to ceftazidime and/or cefotaxime were examined for susceptibility to cefepime. The most frequently encountered ceftazidime-cefotaxime-resistant strains belonged to the genera Enterobacter, Pseudomonas, and Citrobacter. Among these strains, 92% were resistant to cefoperazone, 91% were resistant to cefotaxime, 84% were resistant to ceftazidime, and 6% were resistant to cefepime. Of the members of the family Enterobacteriaceae, 57% were resistant to ceftriaxone. The six strains resistant to cefepime were all Pseudomonas aeruginosa and were resistant to both cefotaxime and ceftazidime. Cefepime-resistant P. aeruginosa strains had exceptionally high levels of beta-lactamase activity, higher than the levels found in strains resistant to ceftazidime but susceptible to cefepime. The beta-lactamases from the cefepime-resistant strains were type I (Richmond-Sykes), were constitutively produced, and did not have increased affinity or hydrolytic activity for cefepime. Thus, cefepime was active against most gram-negative bacteria which have developed resistance to the broad-spectrum cephalosporins, and resistance to cefepime in P. aeruginosa appears to be associated with higher beta-lactamase levels than in cefepime-susceptible strains.     

Antibiotic resistance in staphylococci from a hospital environment

The antibiotic resistance of Staphylococcus aureus and coagulase-negative staphylococci has been followed over 4 years (1977-1980). 90% of the strains produced beta-lactamase. Coagulase-negative staphylococci were more resistant than S. aureus, although beta-lactamase production occurred more rarely. Strains from the sputum of children with cystic fibrosis (CF) were more multiresistant than isolates from other sources. The strains from hospitalized patients (HP) were more resistant than isolates from out-patients. A higher frequency of beta-lactamase production occurred among strains from CF and HP patients compared to out-patients. This occurred in samples from pus and wounds in 66% of the strains derived from non-hospitalized patients compared with 93% from surgical patients.     

Variation in induction of chromosomal beta-lactamase expression in strains of Enterobacter cloacae

Eight strains of Enterobacter cloacae with varying patterns of susceptibility to beta-lactam antibiotics were studied to compare the inducibility and expression of their beta-lactamase genes. These strains included two isolates from one patient, which differed in their susceptibility to cefamandole. A resistant strain constitutively produced large amounts of beta-lactamase; a sensitive strain produced an inducible beta-lactamase. Study of induction and growth characteristics of all strains revealed that the induction and the expression of inducible beta-lactamase genes may vary considerably among strains of E. cloacae.     

Molecular and biochemical characterization of a carbapenem-hydrolysing beta-lactamase from Flavobacterium johnsoniae

Flavobacterium johnsoniae CIP100931 is resistant to most beta-lactam antibiotics and has a decreased susceptibility to carbapenems. A beta-lactamase gene was cloned and expressed in Escherichia coli DH10B. The purified beta-lactamase, JOHN-1, with a pI value of 9.0 and with a determined relative molecular mass of approximately 27 kDa was found to be a monomeric zinc-dependent enzyme that hydrolyses penicillins, narrow- and expanded-spectrum cephalosporins, carbapenems, but not monobactams. Sequence analysis revealed that JOHN-1 is a molecular class B beta-lactamase that is most closely related to BlaB from Chryseobacterium meningosepticum and IND-1 from Chryseobacterium indologenes (47% and 41% amino acid identity, respectively). JOHN-1 is a new member of the highly divergent subclass B1 lineage of metallo-enzymes. Although F. johnsoniae and Chryseobacterium spp. are phylogenetically related bacteria, this report further underlines the heterogeneity of class B beta-lactamases that are naturally produced by environmental Gram-negative aerobes and that are now recognized as the most important reservoir for these beta-lactamase genes.     

Escherichia coli resistant to ampicillin/sulbactam.

Escherichia coli strains resistant to ampicillin/sulbactam from hospitals in 4 different geographic locations were examined with respect to type and amount of beta-lactamase produced. A total of 5 strains was examined from each region. The isoelectric points of all of the involved beta-lactamases were 5.4, corresponding to TEM-1. Km and Vmax values of the beta-lactamases among the clinical isolates resembled those from the control TEM-1 strain. In an 18-hour broth culture the highly resistant isolates produced 3 times more beta-lactamase as compared to the ampicillin/sulbactam-susceptible isolates. However, the highly resistant strains contained approximately the same amount of plasmid DNA (size of > 6,500 bp) as the susceptible isolates. In transformation experiments, both the resistance and the degree of resistance appeared to have been transferable by plasmids. The mechanism for resistance is likely to be a baseline overproduction of TEM-1 beta-lactamase due to either an alteration in the control of gene expression or simply to an increase in the number of copies of the beta-lactamase gene in the plasmids.     

鲍曼不动杆菌对β-内酰胺类抗生素耐药性分析

目的对多重耐药的鲍曼不动杆菌进行β-内酰胺类抗生素耐药性分析。方法K-B法、琼脂稀释法检测35株鲍曼不动杆菌对14种β-内酰胺类抗生素的敏感性;碘—淀粉测定法、三维试验、协同法、纸片扩散确证试验检测青霉素酶、头孢菌素酶(AmpCs)、金属β-内酰胺酶和超广谱β-内酰胺酶(ESBLs);PCR扩增β-内酰胺类抗生素耐药基因Tem-1。结果35株菌株有28株表现为对β-内酰胺类抗生素多重耐药,其中产青霉素酶的菌株16株,产AmpCs的菌株10株,产金属β-内酰胺酶的菌株2株,产ESBLs的菌株3株;9株同时产青霉素酶和AmpCs,2株同时产金属β-内酰胺酶和ESBLs。多数耐药菌株均扩增出Tem-1基因。结论鲍曼不动杆菌产生β-内酰胺酶是其对β-内酰胺类抗生素耐药的主要原因。     

Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC beta-lactamase, and the foss of an outer membrane protein.

Six Escherichia coli and 12 Klebsiella pneumoniae isolates from a single hospital expressed a common beta-lactamase with a pI of approximately 9.0 and were resistant to cefoxitin and cefotetan (MIC ranges, 64 to > 128 and 16 to > 128 micrograms/ml, respectively). Seventeen of the 18 strains produced multiple beta-lactamases. Most significantly, three K. pneumoniae strains were also resistant to imipenem (MICs, 8 to 32 micrograms/ml). Spectrophotometric beta-lactamase assays with purified enzyme indicated hydrolysis of cephamycins, in addition to cephaloridine and benzylpenicillin. The 4ene encoding the pI 9.0 beta-lactamase (designated ACT-1 for AmpC type) was cloned and sequenced, which revealed an ampC-type beta-lactamase gene that originated from Enterobacter cloacae and that had 86% sequence homology to the P99 beta-lactamase and 94% homology to the partial sequence of MIR-1. Southern blotting revealed that the gene encoding ACT-1 was on a large plasmid in some of the K. pneumoniae strains as well as on the chromosomes of all of the strains, suggesting that the gene is located on an easily mobilized element. Outer membrane protein profiles of the K. pneumoniae strains revealed that the three imipenem-resistant strains were lacking a major outer membrane protein of approximately 42 kDa which was present in the imipenem-susceptible strains. ACT-1 is the first plasmid-mediated AmpC-type beta-lactamase derived from Enterobacter which has been completely sequenced. This work demonstrates that in addition to resistance to cephamycins, imipenem resistance can occur in K. pneumoniae when a high level of the ACT-1 beta-lactamase is produced in combination with the loss of a major outer membrane protein.     

Role of OmpD2 and chromosomal beta-lactamase in carbapenem resistance in clinical isolates of Pseudomonas aeruginosa.

Imipenem-resistant clinical isolates of Pseudomonas aeruginosa were divided into two categories: (i) isolates that were moderately resistant to imipenem (MIC 6.25 mg/L) that produced trace amounts of protein D2 detected with immunoblotting using anti-protein D2 antibody, but not when stained with Coomassie blue and had inducible class 1 beta-lactamase expression; (ii) isolates that were highly resistant to several beta-lactams, including meropenem, with no protein D2 by staining or immunoblotting and had stably derepressed beta-lactamase. Laboratory strains were isolated and analyzed: (i) mutants lacking protein D2, or (ii) lacking protein D2 and producing stably derepressed beta-lactamase with carbapenem resistance similar to the clinical isolates. (iii) mutants producing undetectable beta-lactamase which were four-fold more susceptible to imipenem than the mutant producing stably derepressed beta-lactamase or the strain with inducible beta-lactamase. These data suggests that beta-lactamase and outer membrane permeability govern meropenem-resistance in P. aeruginosa.     

Emergence of multidrug-resistant Klebsiella pneumoniae isolates producing VIM-4 metallo-beta-lactamase, CTX-M-15 extended-spectrum beta-lactamase, and CMY-4 AmpC beta-lactamase in a Tunisian university hospital

Klebsiella pneumoniae clinical isolates resistant to carbapenems were recovered from 11 patients in the hospital of Sfax, Tunisia. The isolates were closely related as shown by pulsed-field gel electrophoresis, and they produced VIM-4 metallo-enzyme, CTX-M-15 extended-spectrum beta-lactamase, and CMY-4 AmpC enzyme. The bla(VIM-4) gene is part of a class 1 integron.     

Susceptibility of Ampicillin-Resistant Haemophilus influenzae to Seven Penicillins

Sixty-seven clinical isolates of Haemophilus influenzae from various sections of the United States, England, and Germany were tested for susceptibility to penicillin, ampicillin, amoxicillin, epicillin, carbenicillin, ticarcillin, and methicillin. Fifty-three of the strains had previously been judged to be ampicillin resistant and 14 had been determined to be ampicillin susceptible. Fifty-two of the 53 resistant strains produced beta-lactamase, but none of the susceptible strains produced it. On the basis of minimal inhibitory concentrations, the most active compounds were ticarcillin and carbenicillin. Whether this greater activity is useful clinically has not been established.     

Effect of Inoculum and of Beta-Lactamase on the Anti-Staphylococcal Activity of Thirteen Penicillins and Cephalosporins

Because there are few persuasive data for selecting one semisynthetic penicillin or cephalosporin over another for treatment of serious staphylococcal infections, 118 recent clinical isolates of Staphylococcus aureus were studied to determine to what extent the presence of beta-lactamase affected the relative anti-staphylococcal activity of six penicillins and seven cephalosporins. In addition, the effect of inoculum was studied for its possible effect on the anti-staphylococcal activity of the 13 beta-lactam antibiotics. By all criteria, methicillin and nafcillin were clearly more resistant to both the inoculum effect and the production of staphylococcal beta-lactamase, whereas benzylpenicillin and cephaloridine (especially benzyl-penicillin) were the most susceptible to these effects. Cephazolin was clearly more susceptible to staphylococcal beta-lactamase and heavy inocula than the other cephalosporins (with the exception of cephaloridine), whereas cephalothin was the most resistant cephalosporin to these factors. The minimal inhibitory concentration for benzylpenicillin for tests with undiluted inoculum, compared to results with inoculum diluted 10(-4), differed by a factor up to 16,384, whereas with methicillin and nafcillin the differences were rarely more than twofold. Ratios for the other 10 antibiotics fell between these extremes. These results suggest that methicillin or nafcillin is most stable to staphylococcal beta-lactamase, and that benzylpenicillin and cephaloridine are the most susceptible.     

Differences in the resistant variants of Enterobacter cloacae selected by extended-spectrum cephalosporins.

The rates of development of resistance to ceftriaxone, ceftazidime, cefepime, and cefpirome in 10 strains of Enterobacter cloacae were determined by daily transfer for 7 days to fresh medium containing twofold serial dilutions of the antibiotics. Development of resistance to ceftriaxone was the most rapid; this was followed by ceftazidime, cefpirome, and cefepime. Resistant variants selected by ceftriaxone and ceftazidime were cross-resistant and produced very high levels of beta-lactamase. On the other hand, resistant variants selected by cefepime and cefpirome often had moderately high levels of beta-lactamase and diminished levels of the 39- to 40-kDa porin protein.     

Epidemiology of conjugative plasmid-mediated AmpC beta-lactamases in the United States

A sample of 752 resistant Klebsiella pneumoniae, Klebsiella oxytoca, and Escherichia coli strains from 70 sites in 25 U.S. states and the District of Columbia was examined for transmissibility of resistance to ceftazidime and the nature of the plasmid-mediated beta-lactamase involved. Fifty-nine percent of the K. pneumoniae, 24% of the K. oxytoca, and 44% of the E. coli isolates transferred resistance to ceftazidime. Plasmids encoding AmpC-type beta-lactamase were found in 8.5% of the K. pneumoniae samples, 6.9% of the K. oxytoca samples, and 4% of the E. coli samples, at 20 of the 70 sites and in 10 of the 25 states. ACT-1 beta-lactamase was found at eight sites, four of which were near New York City, where the ACT-1 enzyme was first discovered; ACT-1 beta-lactamase was also found in Massachusetts, Pennsylvania, and Virginia. FOX-5 beta-lactamase was also found at eight sites, mainly in southeastern states but also in New York. Two E. coli strains produced CMY-2, and one K. pneumoniae strain produced DHA-1 beta-lactamase. Pulsed-field gel electrophoresis and plasmid analysis suggested that AmpC-mediated resistance spread both by strain and plasmid dissemination. All AmpC beta-lactamase-containing isolates were resistant to cefoxitin, but so were 11% of strains containing transmissible SHV- and TEM-type extended-spectrum beta-lactamases. A beta-lactamase inhibitor test was helpful in distinguishing the two types of resistance but was not definitive since 24% of clinical isolates producing AmpC beta-lactamase had a positive response to clavulanic acid. Coexistence of AmpC and extended-spectrum beta-lactamases was the main reason for these discrepancies. Plasmid-mediated AmpC-type enzymes are thus responsible for an appreciable fraction of resistance in clinical isolates of Klebsiella spp. and E. coli, are disseminated around the United States, and are not so easily distinguished from other enzymes that mediate resistance to oxyimino-beta-lactams.     

Selection frequency of resistant variants by various beta-lactam antibiotics in clinical Enterobacter cloacae isolates

The frequency of selection of resistant variants by 10 different broad-spectrum beta-lactam derivatives was evaluated for 10 clinical Enterobacter cloacae isolates. With respect to most penams or cephems, resistant variants could be selected up to 8- or 32-fold the MIC, respectively. However, with cefpirome as the selecting agent resistant variants were obtained only at twice the MIC, whereas resistant variants were barely detectable with temocillin and not detectable in any case with imipenem. The variants exhibited cross-resistance between penams and cephems including aztreonam, but not to temocillin and imipenem regardless of the beta-lactamase amount produced. Enzyme production of the variants ranged from 0.2 U to 19.0 U beta-lactamase/mg protein of the cell-free supernatants. Moreover, analysis of the outer membrane protein composition did not reveal marked alterations between wild strains and the corresponding variants. It is evident that 'overproduction of the chromosomal beta-lactamase' cannot explain entirely phenotypic resistance to broad-spectrum beta-lactam compounds, but a lack of porin production, i.e., major outer membrane proteins, cannot provide an explanation for the above findings.     

Detection of OXA-4 beta-lactamase in Pseudomonas aeruginosa isolates by genetic methods

In Pseudomonas aeruginosa, resistance to cefclidin is usually associated with resistance to another third-generation cephalosporin, ceftazidime. In this study we analysed 22 isolates of P. aeruginosa, collected at Showa University Fujigaoka Hospital between 1992 and 1993, which were resistant to cefclidin but susceptible to ceftazidime. All polymerase chain reaction (PCR) products amplified by a primer pair covering the full-length gene of OXA-4 (also OXA-1) precursor beta-lactamase were 0.84 kb in length. The isoelectric points of the beta-lactamases produced by these isolates were typical of the OXA-4 type of beta-lactamase (pl 7.5) rather than the OXA-1 type (pl 7.4). All PCR products at 216 bp were amplified by the primer pair covering the A928-->T point mutation, which corresponds to the Asp48-->Val amino acid substitution of OXA-1 beta-lactamase to form OXA-4 beta-lactamase. These single-strand conformation polymorphism (SSCP) patterns are typical of the OXA-4 gene, rather than the OXA-1 gene, demonstrating that these enzymes can be classified by SSCP analyses based on the PCR method. Although OXA-4 beta-lactamase is generally plasmid-mediated, the chromosomal DNA of these isolates, but not their plasmids, hybridized with the OXA-4 gene amplified by the PCR method. Based on these results, we suspected that the plasmids encoding OXA-4 beta-lactamase had been spontaneously cured, or that the gene had been deleted from the plasmid. The distribution of P. aeruginosa producing OXA-4 beta-lactamase amongst hospital wards and clinical specimens demonstrated that the OXA-4 enzyme in this collection period was representative of hospital P. aeruginosa.     

Selection of resistance by piperacillin during Pseudomonas aeruginosa endocarditis

The mechanisms responsible for emergence of resistance during antimicrobial therapy were investigated in isolates obtained from a patient suffering from Pseudomonas aeruginosa endocarditis. The strain was first isolated from blood cultures obtained upon admission of the patient. It was sensitive to piperacillin, ticarcillin and tobramycin. Piperacillin-tobramycin therapy was therefore instituted and led to a rapid improvement of the patient's condition. Unfortunately, the patient subsequently became febrile again and blood cultures continued to yield P. aeruginosa. However, the organism isolated was now resistant to piperacillin and other penicillins tested. Cell-free extracts of the pre- and post-therapy isolates were analyzed for their beta-lactamase activity. The susceptible pre-therapy strain did not produce significant levels of beta-lactamase. In contrast, the post-therapy strain produced high levels of the enzyme. Induction experiments indicated that the production of beta-lactamase was constitutive in the post-therapy isolate. Thus, piperacillin therapy has led to the selection of resistant mutants which produced high levels of beta-lactamase constitutively. This was associated with relapse of the infection and therapeutic failure.     

National collaborative study of the prevalence of antimicrobial resistance among clinical isolates of Haemophilus influenzae.

A total of 2,811 clinical isolates of Haemophilus influenzae were obtained during 1986 from 30 medical centers and one nationwide private independent laboratory in the United States. Among these, 757 (26.9%) were type b strains. The overall rate of beta-lactamase-mediated ampicillin resistance was 20.0%. Type b strains were approximately twice as likely as non-type b strains to produce beta-lactamase (31.7 versus 15.6%). The MICs of 12 antimicrobial agents were determined for all isolates. Ampicillin resistance among strains that lacked beta-lactamase activity was extremely uncommon (0.1%). Percentages of study isolates susceptible to cefamandole, cefaclor, cephalothin, and cephalexin were 98.7, 94.5, 87.3, and 43.3%, respectively. For 14 strains (0.5% of the total), chloramphenicol MICs were greater than or equal to 8.0 micrograms, and thus the strains were considered resistant. All of these resistant strains produced chloramphenicol acetyltransferase. In addition, all 14 strains were resistant to tetracycline; 11 produced beta-lactamase. The percentage of isolates susceptible to tetracycline was 97.7%. In contrast, erythromycin and sulfisoxazole were relatively inactive. The combination of erythromycin-sulfisoxazole (1/64) was more active than erythromycin alone but essentially equivalent in activity to sulfisoxazole alone. Finally, small numbers of clinical isolates of H. influenzae were resistant to trimethoprim-sulfamethoxazole and rifampin.