Invasion of human fallopian tube epithelium by Escherichia coli expressing combinations of a gonococcal porin, opacity-associated protein, and chimeric lipo-oligosaccharide.

The transepithelial migration of Escherichia coli that expressed all possible combinations of a plasmid-encoded gonococcal porin (Por), opacity-associated protein (Opa), and 3F11 lipo-oligosaccharide (LOS) epitope was investigated. Surface expression of Por mediated selective changes in E. coli antibiotic susceptibility, and coexpression of Opa and the 3F11 LOS epitope mediated bacterial clumping (P<.01). In the human fallopian tube organ-culture model, Opa-producing variants attached up to 44-fold better than control bacteria (P<.01), and Por-producing variants exceeded submucosal invasion of control bacteria by 500-fold (P<.01). Opa and Por each facilitated intracellular invasion 20-40-fold (P<.01). In dual expresser variants, the 3F11 LOS epitope markedly reduced attachment and invasion mediated by Opa or Por. The LOS inhibitory effect was curbed when all 3 factors were expressed, which suggests an additional interaction of the 3 factors at the bacterial surface. Por, Opa, and LOS play important roles in Neisseria gonorrhoeae trafficking across human fallopian tube epithelium.

Frequency of pathogen occurrence and antimicrobial susceptibility among community-acquired respiratory tract infections in the respiratory surveillance program study: microbiology from the medical office practice environment.

Continuing problems of antimicrobial resistance have prompted the initiation of several surveillance programs. Few, if any, of these programs focus on community-acquired respiratory tract infections seen in routine office-based practices. The Respiratory Surveillance Program (RESP; 1999-2000) in 674 community-based physician office practices in the United States determined the frequency of potential bacterial pathogens including Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in patients diagnosed clinically with community-acquired pneumonia, acute exacerbations of chronic bronchitis, and sinusitis throughout all 9 US census/geographic regions. Susceptibility to the penicillins (ampicillin, penicillin), oral cephalosporins, fluoroquinolones (gatifloxacin, levofloxacin, ciprofloxacin), macrolides (erythromycin, azithromycin, clarithromycin), tetracycline, and trimethoprim/sulfamethoxazole was determined by reference methods. Patients were required to have a culturable focus of infection, and specimens were immediately sent to a reference laboratory. Among 22,689 total specimens (610 community-acquired pneumonia, 4,779 acute exacerbation of chronic bronchitis, 16,213 sinusitis, 1,087 other), H influenzae was the most commonly isolated organism from patients with community-acquired pneumonia (38%) and acute exacerbation of chronic bronchitis (35%) in all nine geographic regions. S pneumoniae was isolated in 18% of community-acquired pneumonia cases, 13% of acute exacerbation of chronic bronchitis cases, and 11% of sinusitis cases. M catarrhalis was most commonly isolated from the nasopharynx of patients with sinusitis (29%). High-level resistance to penicillin (2 microg/mL or greater; 16% overall) and the macrolides (32% to 35%) among S pneumoniae varied both with site of infection and with geographic region. The greatest resistance was observed among isolates from the nasopharynx of patients with sinusitis and from patients from the East South Central or South Atlantic regions of the United States. Although the susceptibility of H influenzae and M catarrhalis to the tested antimicrobials did not vary with the type of infection, beta-lactamase-mediated resistance to ampicillin among H influenzae ranged from 15% in New England to 32% in the East South Central region. The fluoroquinolones were highly active against these cultured isolates from community-acquired respiratory tract infection patients, with >99% of all S pneumoniae, H influenzae, and M catarrhalis strains susceptible to gatifloxacin (MIC(90), 0.5 microg/mL) and levofloxacin (MIC(90), 2 microg/mL). The extended-spectrum fluoroquinolones appear well suited for community-acquired respiratory tract infection therapy, including pathogens other than pneumococcus, H influenzae, and M catarrhalis.

Clinical resistance encountered in the respiratory surveillance program (RESP) study: a review of the implications for the treatment of community-acquired respiratory tract infections.

The Respiratory Surveillance Program (RESP) is a large-scale surveillance study of potential bacterial pathogens from respiratory tract infections that was performed over a 10-month period (July to April) during the 1999-2000 respiratory infection season. It is also the first study of its kind to derive its information entirely from community-based medical practices. This study, therefore, provides insight into the identity, frequency, and susceptibility of the possible pathogens isolated from patients encountered by primary care physicians. Reduction of antibiotic susceptibility in various bacterial pathogens may be of academic interest. However, it is only the emergence of clinical resistance (strains exhibiting minimum inhibitory concentrations above the resistance breakpoint) to commonly used antibacterial agents in the most prevalent species that has significant impact on empiric therapy choices. A review of data from RESP indicated that the most prevalent species were Moraxella catarrhalis, Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae. As expected, the prevalence of these bacterial isolates varied by disease state. The prevalence of clinical resistance to various antibiotics ranged, within these 4 species, between 0% and 92%. Resistance to the greatest number of drugs was expressed by S pneumoniae, followed by S aureus, H influenzae, and M catarrhalis. The prevalence of antibiotic resistance found among these community-isolated pathogens was surprisingly similar to that reported in hospital-based studies, suggesting that resistance is as important an issue in the community as it is in hospitals. With few exceptions, the prevalence of resistance was fairly uniform across disease states. The antibiotics most likely to encounter clinically resistant isolates during the treatment of community-acquired respiratory tract infections were penicillins, macrolides, and trimethoprim/sulfamethoxazole. The antibiotics least likely to encounter resistance were quinolones, followed by ceftriaxone and amoxicillin/clavulanate.

Use of an isogenic Escherichia coli panel to design tests for discrimination of beta-lactamase functional groups of Enterobacteriaceae.

A study was designed to determine if an isogenic panel of Escherichia coli strains containing many different beta-lactamases could be used for the preliminary screening of a large number of beta-lactam agents to identify which might be most useful in the development of a definitive test for specific beta-lactamases found among the members of family Enterobacteriaceae. The susceptibilities of 46 strains, comprising the isogenic panel, to expanded-spectrum cephalosporins, cephamycins, and aztreonam were determined in the presence and absence of beta-lactamase inhibitors in broth microdilution tests. The results indicated that strains producing extended-spectrum beta-lactamases (ESBLs) could be distinguished from strains producing other Bush-Jacoby-Medeiros functional group 2 or group 1 beta-lactamases. For strains producing group 1 beta-lactamases, cefpodoxime and ceftazidime MICs were > or = 4 micrograms/ml and addition of clavulanate did not reduce the MICs more than fourfold. For strains producing group 2 enzymes other than ESBLs, cefpodoxime and ceftazidime MICs were < or = 2 micrograms/ml. With a single exception (ceftazidime for the strain producing SHV-3), among strains producing ESBLs, cefpodoxime and ceftazidime MICs were > or = 4 micrograms/ml and addition of clavulanate reduced the MICs by more than eightfold. Cephamycins could also be used to discriminate between strains producing group 1 beta-lactamases and ESBLs, since only the former required cefotetan concentrations as high as 8 micrograms/ml or cefoxitin concentrations of > 16 micrograms/ml for inhibition. Other cephalosporins provided some discrimination between the various beta-lactamase producers, although they were not as reliable as either cefpodoxime or ceftazidime. These results indicate the utility of an isogenic panel for identification of candidate drugs among many for further testing with clinical isolates of the family Enterobacteriaceae to determine the best agents for detection of specific beta-lactamases in this family.

beta-Lactamases responsible for resistance to expanded-spectrum cephalosporins in Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis isolates recovered in South Africa.

Although resistance to the expanded-spectrum cephalosporins among members of the family Enterobacteriaceae lacking inducible beta-lactamases occurs virtually worldwide, little is known about this problem among isolates recovered in South Africa. Isolates of Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis resistant to expanded-spectrum cephalosporins recovered from patients in various parts of South Africa over a 3-month period were investigated for extended-spectrum beta-lactamase production. Antibiotic susceptibility was determined by standard disk diffusion and agar dilution procedures. Production of extended-spectrum beta-lactamases was evaluated by using the double-disk test, and the beta-lactamases were characterized by spectrophotometric hydrolysis assays and an isoelectric focusing overlay technique which simultaneously determined isoelectric points and general substrate or inhibitor characteristics. DNA amplification and sequencing were performed to confirm the identities of these enzymes. The P. mirabilis and E. coli isolates were found to produce TEM-26-type, SHV-2, and SHV-5 extended-spectrum beta-lactamases. An AmpC-related enzyme which had a pI of 8.0 and which conferred resistance to cefoxitin as well as the expanded-spectrum cephalosporins was found in a strain of K. pneumoniae. This is the first study which has identified organisms producing different extended-spectrum beta-lactamases from South Africa and the first report describing strains of P. mirabilis producing a TEM-26-type enzyme. The variety of extended-spectrum beta-lactamases found among members of the family Enterobacteriaceae isolated from major medical centers in South Africa is troubling and adds to the growing list of countries where these enzymes pose a serious problem for antimicrobial therapy.

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

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

Structure-activity studies of quinolone-penems in genetically defined strains of Escherichia coli.

Quinolonyl-beta-lactam antimicrobial agents (QLAs) contain quinolones chemically linked to beta-lactams, although the impact of linkage is poorly understood. Genetically defined Escherichia coli strains were used to determine structure-activity characteristics of three quinolone-penem QLAs. Results suggest that the leaving group resulting from beta-lactam hydrolysis may not be free quinolone.

Penicillin-binding proteins and induction of AmpC beta-lactamase.

In competition assays for radiolabeled penicillin, penicillin-binding proteins (PBPs) 4, 7a, and 7b showed very high affinities for strong inducers of AmpC beta-lactamase. Loss of PBP 4 resulted in diminished inducibility. This suggests that if PBPs are involved in induction of AmpC beta-lactamase, there is probably a redundancy in function among the different PBPs.

Sequencing and analysis of four new Enterobacter ampD Alleles.

Sequences of ampD genes from wild-type, temperature-sensitive, and stably derepressed mutants of the wild-type strain of Enterobacter cloacae 029 and the hyperinducible strain E. cloacae 1194E were determined and compared with the ampD gene of the wild-type strain E. cloacae 14. Seventy nucleotide differences were found between the wild-type sequences, resulting in 13 amino acid changes. The deduced amino acid changes do not correspond to published AmpC regulation mutations and expand the number of known mutations leading to altered AmpC beta-lactamase expression in members of the family Enterobacteriaceae.

beta-Lactam resistance amongst Enterobacter species.

Following the introduction of extended-spectrum cephalosporins into clinical use, the prevalence of species belonging to the genus Enterobacter has increased because of their natural resistance to earlier cephalosporins and their ability to develop resistance rapidly to the newer drugs. beta-Lactam resistance in this genus is due, for the most part, to the presence of a Bush group 1 chromosomal cephalosporinase. This enzyme is normally inducible and resistance to older cephalosporins, cephamycins and aminopenicillins results from either the extreme lability of the drugs to the enzyme or from their inducer activities. Resistance to newer penicillins, cephalosporins and monobactams is attributable to the selection of mutants which express large amounts of the enzyme. Such mutants arise as the result of a spontaneous mutation in one of the regulatory genes responsible for suppressing enzyme expression. Since the enzyme has very high affinity for the newer cephalosporins, this, coupled with the slow penetration of the drugs into the cell, provides a very efficient mechanism of resistance. Recent surveys in the USA and elsewhere have shown that the increased prevalence of multi-beta-lactam-resistant strains of enterobacter is due to the increased use of the newer cephalosporins. Attempts to prevent these problems include the more judicious use of newer beta-lactam antibiotics and the development of enhanced-potency cephalosporins which are able to avoid resistance because they have lower enzyme affinity and permeate more rapidly into the cell.

Emergence of resistance to imipenem in Enterobacter isolates masquerading as Klebsiella pneumoniae during therapy with imipenem/cilastatin.

Clinical isolates identified as Klebsiella pneumoniae by the Vitek, Enterotube II, and API 20E systems were recovered from a patient undergoing therapy with imipenem/cilastatin. These isolates were resistant to multiple beta-lactam agents, and some were even resistant to imipenem. Analysis revealed a Bush group 1 beta-lactamase, and imipenem resistance corresponded to the loss of outer-membrane proteins in strains expressing high levels of this beta-lactamase. Further characterization efforts yielded abnormal but positive results of tests for ornithine decarboxylase production and motility, and chromosomal homology to an Enterobacter cloacae ampR, ampC probe was detected. These results suggested that the organisms were actually of an Enterobacter species, perhaps Enterobacter aerogenes. Cefoxitin resistance may be a useful marker for preventing this misidentification in the future; misidentification of such organisms poses a hazard, as it may lead to inappropriate beta-lactam therapy for infections caused by organisms that have the potential for resistance due to inducible group 1 cephalosporinases.

Genetic determinants of host ranges of Bacillus sphaericus mosquito larvicidal toxins.

The 51.4-kDa-41.9-kDa binary toxin produced by different strains of Bacillus sphaericus shows differential activity toward Culex quinquefasciatus, Aedes atropalpus, and Aedes aegypti mosquito larvae. The patterns of larvicidal activity toward all three mosquito species and growth retardation in A. aegypti have been shown to be due to the 41.9-kDa protein. By using mutant toxins expressed in Escherichia coli, insecticidal activity and growth retardation correlated with amino acids centered around position 100 of the 41.9-kDa protein. In its response to these toxins, A. atropalpus resembled C. quinquefasciatus rather than its congener, A. aegypti.