Multiple Low-Reactivity Class B Penicillin-Binding Proteins Are Required for Cephalosporin Resistance in Enterococci.

Enterococcus faecalis and Enterococcus faecium are commensals of the gastrointestinal tract of most terrestrial organisms, including humans, and are major causes of health care-associated infections. Such infections are difficult or impossible to treat, as the enterococcal strains responsible are often resistant to multiple antibiotics. One intrinsic resistance trait that is conserved among E. faecalis and E. faecium is cephalosporin resistance, and prior exposure to cephalosporins is one of the most well-known risk factors for acquisition of an enterococcal infection. Cephalosporins inhibit peptidoglycan biosynthesis by acylating the active-site serine of penicillin-binding proteins (PBPs) to prevent the PBPs from catalyzing cross-linking during peptidoglycan synthesis. For decades, a specific PBP (known as Pbp4 or Pbp5) that exhibits low reactivity toward cephalosporins has been thought to be the primary PBP required for cephalosporin resistance. We analyzed other PBPs and report that in both E. faecalis and E. faecium, a second PBP, PbpA(2b), is also required for resistance; notably, the cephalosporin ceftriaxone exhibits a lethal effect on the ΔpbpA mutant. Strikingly, PbpA(2b) exhibits low intrinsic reactivity with cephalosporins in vivo and in vitro Unlike the Δpbp5 mutant, the ΔpbpA mutant exhibits a variety of phenotypic defects in growth kinetics, cell wall integrity, and cellular morphology, indicating that PbpA(2b) and Pbp5(4) are not functionally redundant and that PbpA(2b) plays a more central role in peptidoglycan synthesis. Collectively, our results shift the current understanding of enterococcal cephalosporin resistance and suggest a model in which PbpA(2b) and Pbp5(4) cooperate to coordinately mediate peptidoglycan cross-linking in the presence of cephalosporins.

Experimental study of meropenem in the therapy of cephalosporin-susceptible and -resistant pneumococcal meningitis.

Meropenem is a carbapenem antibiotic that is highly active against the pathogens causing meningitis. Results with meropenem in the experimental rabbit model of penumococcal meningitis have been controversial, and the possible role of renal dehydropeptidase I in meropenem efficacy has been suggested. The aim of this study was to determine the efficacy of meropenem in two meningitis models and the possible influence of the animal model over results. Two strains of Streptococcus pneumoniae with different susceptibility to beta-lactams have been used in a guinea pig model and the classical rabbit meningitis model. Meropenem was bactericidal at 6 h in the guinea pig model against both strains with a reduction of >4 log ufc/ml. In the rabbit model it was bactericidal at 6 h against the susceptible strain, but against the resistant 3/8 therapeutical failures were recorded at 6 h, being bactericidal at 24 h. In conclusion, meropenem has shown bactericidal activity in both experimental models. This work emphasises the importance of an adequate election of the animal model for the appropriate development of studies of antimicrobial efficacy. We believe that guinea pig should be considered the best choice among laboratory animal species when assessing meropenem efficacy.

Mechanisms of antimicrobial resistance in bacteria.

The treatment of bacterial infections is increasingly complicated by the ability of bacteria to develop resistance to antimicrobial agents. Antimicrobial agents are often categorized according to their principal mechanism of action. Mechanisms include interference with cell wall synthesis (eg, beta-lactams and glycopeptide agents), inhibition of protein synthesis (macrolides and tetracyclines), interference with nucleic acid synthesis (fluoroquinolones and rifampin), inhibition of a metabolic pathway (trimethoprim-sulfamethoxazole), and disruption of bacterial membrane structure (polymyxins and daptomycin). Bacteria may be intrinsically resistant to > or =1 class of antimicrobial agents, or may acquire resistance by de novo mutation or via the acquisition of resistance genes from other organisms. Acquired resistance genes may enable a bacterium to produce enzymes that destroy the antibacterial drug, to express efflux systems that prevent the drug from reaching its intracellular target, to modify the drug's target site, or to produce an alternative metabolic pathway that bypasses the action of the drug. Acquisition of new genetic material by antimicrobial-susceptible bacteria from resistant strains of bacteria may occur through conjugation, transformation, or transduction, with transposons often facilitating the incorporation of the multiple resistance genes into the host's genome or plasmids. Use of antibacterial agents creates selective pressure for the emergence of resistant strains. Herein 3 case histories-one involving Escherichia coli resistance to third-generation cephalosporins, another focusing on the emergence of vancomycin-resistant Staphylococcus aureus, and a third detailing multidrug resistance in Pseudomonas aeruginosa-are reviewed to illustrate the varied ways in which resistant bacteria develop.

Enterobacter meningitis: organism susceptibilities, antimicrobial therapy and related outcomes.

BACKGROUND: Meningitis due to Enterobacter species is an uncommon infection in adults; however, when present, treatment is frequently complicated by resistance of many Enterobacter isolates to third-generation cephalosporins and poor central nervous system penetration of other antibiotics. The aim of this study was to retrospectively review cases of meningitis caused by Enterobacter species at our institution, to better characterize patient factors, pathogen characteristics, and treatment options for this infection. METHODS: We reviewed all cases of Enterobacter meningitis in a 12-year period at a tertiary care center. Data collected included patient demographics, antibiotic sensitivities of Enterobacter isolates, antimicrobial therapy, and patient outcomes. RESULTS: Nineteen cases were identified, primarily in patients with neurotrauma and in neurosurgical patients. Enterobacter cloacae was the most frequent Enterobacter species isolated followed by Enterobacter aerogenes and Enterobacter agglomerans (50%, 34%, and 16% of cultures, respectively). Overall, clinical cure/improvement was achieved in 47% of patients, and the mortality rate was 21%. Antibiotic treatment varied substantially and included third-generation cephalosporins, intravenous and intrathecal aminoglycosides, trimethoprim-sulfamethoxazole (TMP-SMX), piperacillin, ciprofloxacin, and other miscellaneous antibiotics. Treatment with TMP-SMX was associated with a high rate of clinical cure/improvement, whereas third-generation cephalosporins were less efficacious. CONCLUSIONS: Enterobacter meningitis is an infrequent complication of neurological insult. Treatment is often complicated by resistance of Enterobacter species to third-generation cephalosporins. Our results indicate that while third-generation cephalosporins are not the most appropriate choice of agents to treat Enterobacter meningitis, TMP-SMX may yield satisfactory results.

Altered PBP 2A and its role in the development of penicillin, cefotaxime, and ceftriaxone resistance in a clinical isolate of Streptococcus pneumoniae.

We report the unusual involvement of altered PBP 2A in the development of beta-lactam resistance in Streptococcus pneumoniae. This was investigated amid three identical serotype 14 isolates (designated isolates 1, 2, and 3, respectively) of pneumococci cultured successfully from the blood of a human immunodeficiency virus-seropositive child with recurrent pneumonia. The passage of this strain through its human host induced several changes in the bacterium, which is typical of the adaptive and evolving nature of the pneumococcus. An efflux resistance mechanism, which conferred increased ciprofloxacin resistance, was induced in isolates 2 and 3. In addition, faster growth rates and larger capsules were also observed for these isolates, with respect to isolate 1. Notably, compared to isolates 1 and 2, isolate 3 showed a decrease in penicillin, cefotaxime, and ceftriaxone resistance. This change was associated with the replacement of an altered PBP 2A for an unaltered PBP 2A. In all likelihood, these events produced a strain which evolved into a fitter and more virulent type, isolate 3, that resulted in an aggravated pneumococcal infection and ultimately in the patient's death.

PER-1- and OXA-10-like beta-lactamases in ceftazidime-resistant Pseudomonas aeruginosa isolates from intensive care unit patients in Istanbul, Turkey.

The presence of PER-1- and OXA-10-like beta-lactamases was investigated by PCR in 49 ceftazidime-resistant Pseudomonas aeruginosa isolates from patients hospitalised in the 24-bed general intensive care unit of the Istanbul Faculty of Medicine during a 12-month period between February 1999 and February 2000. The clonal relatedness of the isolates was investigated by random amplified polymorphic DNA (RAPD) analysis, and the sequences of the PER-1 and OXA genes from all isolates were determined. The rates of resistance of the isolates to imipenem, aztreonam and cefepime were 98%, 92% and 96%, respectively, and to piperacillin and piperacillin-tazobactam were 41% and 37%, respectively. Using the double-disk synergy test, 37% (18/49) of the isolates were identified as extended-spectrum beta-lactamase producers. The PER-1 gene was identified in 86% (42/49) and the OXA-10 gene in 55% (27/49) of the ceftazidime-resistant isolates. Of isolates carrying the PER-1 gene, 48% (20/42) also carried the OXA-10 gene. The respective nucleotide sequences were identical for each isolate. Sixteen RAPD patterns were detected among the PER-1-positive isolates, but 60% (25/42) of the PER-1-positive isolates belonged to two distinct patterns, while the remainder exhibited a wide clonal diversity. The results indicated that the prevalence of PER-1- and OXA-10-like beta-lactamases remains high among ceftazidime-resistant P. aeruginosa isolates in Turkey.

Extended-spectrum-cephalosporin resistance in Salmonella enterica isolates of animal origin.

A total of 112 out of 5,709 Salmonella enterica isolates from domestic animal species exhibited decreased susceptibilities to ceftiofur and ceftriaxone, and each possessed the blaCMY gene. Ten Salmonella serotypes were significantly more likely to include resistant isolates. Isolates from turkeys, horses, cats, and dogs were significantly more likely to include resistant isolates.

Cephalosporin resistant urinary tract infections in young children.

OBJECTIVE: To describe the antibiotic resistance pattern of bacteria causing urinary tract infection (UTI) in a cohort of Australian children under 6 years of age. METHODS: Data were collected over a 12-month period from children under 6 years of age with a provisional diagnosis of UTI made in the Emergency Department of Sunshine Hospital, Victoria. RESULTS: During the study period 100 culture-proven UTI were identified in 97 children. Three children had two episodes. Out of the 100 episodes, 39% were male, 56% were under 12 months of age at presentation and 61% were managed as outpatients. Clinical features were non-specific in the majority of cases. Hydronephrosis and vesicoureteric reflux was detected in 5.5% and 28.6%, respectively, of children with their first investigated UTI. A single bacterial isolate was cultured from 97 urines and two from three samples. Escherichia coli (n = 90) and Proteus mirabilis (n = 5) were the most common isolates. In vitro resistance to ampicillin/amoxycillin was found in 52% of isolates, to trimethoprim in 14% and to cephalothin/cephalexin in 24%. This resistance rate to first generation cephalosporins is the highest reported to date in adult or paediatric UTI in Australia. CONCLUSIONS: Ampicillin/amoxycillin or cephalothin/cephalexin may not be the optimal choice of antibiotic for the empiric treatment of UTI in this and possibly other paediatric populations.

Experimental prediction of the evolution of cefepime resistance from the CMY-2 AmpC beta-lactamase.

Understanding of the evolutionary histories of many genes has not yet allowed us to predict the evolutionary potential of those genes. Intuition suggests that current biochemical activity of gene products should be a good predictor of the potential to evolve related activities; however, we have little evidence to support that intuition. Here we use our in vitro evolution method to evaluate biochemical activity as a predictor of future evolutionary potential. Neither the class C Citrobacter freundii CMY-2 AmpC beta-lactamase nor the class A TEM-1 beta-lactamase confer resistance to the beta-lactam antibiotic cefepime, nor do any of the naturally occurring alleles descended from them. However, the CMY-2 AmpC enzyme and some alleles descended from TEM-1 confer high-level resistance to the structurally similar ceftazidime. On the basis of the comparison of TEM-1 and CMY-2, we asked whether biochemical activity is a good predictor of the evolutionary potential of an enzyme. If it is, then CMY-2 should be more able than the TEMs to evolve the ability to confer higher levels of cefepime resistance. Although we generated CMY-2 evolvants that conferred increased cefepime resistance, we did not recover any CMY-2 evolvants that conferred resistance levels as high as the best cefepime-resistant TEM alleles.

Effect of extended-spectrum beta-lactamases on the susceptibility of Haemophilus influenzae to cephalosporins.

The extended-spectrum beta-lactamases (ESBLs) TEM-3, TEM-4 and TEM-5 were cloned into Haemophilus influenzae. These recombinant strains exhibited cefotaxime MICs of 0.5, 0.25 and 0.12 mg/L for TEM-3, -4 and -5, respectively, and the MIC of cefaclor was 4.0 mg/L. These MICs are higher than those of beta-lactamase-negative strains, or those producing simple wild-type TEM-1 beta-lactamase, but not high enough to be categorized as resistant according to the breakpoints of the NCCLS. The clones were also categorized as susceptible using NCCLS disc diffusion methodology and interpretive criteria. This study shows that current NCCLS susceptibility testing methods may have difficulty in detecting ESBLs if they were to occur in H. influenzae.

Regional occurrence of plasmid-mediated SHV-7, an extended-spectrum beta-lactamase, in Enterobacter cloacae in Philadelphia Teaching Hospitals.

Although the production of extended-spectrum beta-lactamases (ESBLs) by Klebsiella pneumoniae and Escherichia coli is an emerging problem, limited data are available regarding the frequency of ESBL production in other organisms. We provide the only description of regional occurrence of SHV-7 in Enterobacteriaceae other than E. coli or K. pneumoniae in the United States, and we emphasize that, among Enterobacter cloacae strains, not all resistance to extended-spectrum cephalosporins is the result of hyperproduction of AmpC beta-lactamase.

Beta-lactamases involved in resistance to broad-spectrum cephalosporins in Escherichia coli and Klebsiella spp. clinical isolates collected between 1994 and 1996, in Barcelona (Spain).

The aim of this study was to evaluate the incidence of decreased susceptibility to broad-spectrum cephalosporins in Enterobacteriaceae that lack inducible chromosomal bla genes, and to determine the enzymes responsible for resistance. From all clinically relevant Enterobacteriaceae strains isolated between 1994 and 1996, 88 of 7054 Escherichia coli, seven of 581 Klebsiella pneumoniae and 23 of 166 Klebsiella oxytoca strains were studied because of their decreased susceptibilities to broad-spectrum cephalosporins (as reflected in intermediate susceptibilities and/or positive synergy tests and/or irregular crenellated inhibition zones). The most frequent mechanism implicated in decreased susceptibility to broad-spectrum cephalosporins displayed by E. coli and K. oxytoca was hyperproduction of chromosomal beta-lactamase, followed by plasmid-mediated SHV-1 hyperproduction in E. coli. In our hospital, the incidence of plasmid-mediated extended-spectrum beta-lactamases (ESBLs) between 1994 and 1996 was low. ESBLs were found in only 10 (0.14%) E. coli strains (six CTX-M-9, two TEM-12 and two SHV-2), in one (0.17%) K. pneumoniae strain (SHV-2) and in no K. oxytoca strains. The relatively wide variety of beta-lactamases that were detected among these common bacteria isolated from a single medical centre, including non-TEM- and non-SHV-derived ESBLs, appears epidemiologically remarkable.

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).

Risk factors for emergence of resistance to broad-spectrum cephalosporins among Enterobacter spp.

Among 477 patients with susceptible Enterobacter spp., 49 subsequently harbored third-generation cephalosporin-resistant Enterobacter spp. Broad-spectrum cephalosporins were independent risk factors for resistance (relative risk [OR] = 2.3, P = 0.01); quinolone therapy was protective (OR = 0.4, P = 0.03). There were trends toward decreased risk for resistance among patients receiving broad-spectrum cephalosporins and either aminoglycosides or imipenem. Of the patients receiving broad-spectrum cephalosporins, 19% developed resistance.

Antimicrobial susceptibility of Staphylococcus spp. isolated from mammary parenchymas of slaughtered dairy cows.

The high prevalence of the great resistance to antimicrobials shown by the aetiological agents of infectious bovine mastitis, often leads to chronic recurrent mastitis, one of the main causes of early culling of dairy cows. The purpose of this study was to determine the in vitro susceptibility pattern of Staphylococcus spp. isolated from mammary parenchymas of slaughtered dairy cows, to different anti-microbials. A total of 45 Staphylococcus spp. strains [33 coagulase-negative Staphylococcus (CNS) and 12 Staphylococcus aureus (SA)] were used. These strains were tested with 12 different antimicrobials by the Kirby and Bauer standardized disc diffusion method; 84.44% and 86.66% of the 45 strains were resistant to ampicillin and penicillin, respectively. The highest sensitivity was to cephalothin (84.44%), gentamicin (80%) and to sulphazotrin (77.77%). CNS showed higher resistance (P < 0.05) than SA. The in vitro susceptibility pattern of Staphylococcus spp. strains isolated from mammary parenchymas studied in the present investigation was similar to that observed in recent studies of in vitro and in vivo susceptibility patterns of Staphylococcus spp. isolated from cases of bovine mastitis.

Killing activities of trovafloxacin alone and in combination with beta-lactam agents, rifampin, or vancomycin against Streptococcus pneumoniae isolates with various susceptibilities to extended-spectrum cephalosporins at concentrations clinically achievable in cerebrospinal fluid.

The killing activities of trovafloxacin alone and in combination with beta-lactam agents (extended-spectrum cephalosporins, meropenem), rifampin, or vancomycin were evaluated against 20 genotypically characterized Streptococcus pneumoniae isolates for which amoxicillin MICs were >/=4 microg/ml (cefotaxime MICs, >/=4 microg/ml for six strains) at concentrations clinically achievable in cerebrospinal fluid. At 6 h the mean killing activity of trovafloxacin alone (range, 2.6 to 2.9 log(10) CFU/ml) did not vary significantly according to the susceptibility of the strains to beta-lactam agents. The activities of trovafloxacin or vancomycin added to the beta-lactam agents and the combination trovafloxacin-vancomycin were additive or indifferent. Against the ceftriaxone-resistant isolates, the killing activity of the combination of a beta-lactam agent and trovafloxacin did not differ significantly from that of a beta-lactam agent and vancomycin.

Pharmacodynamics of trovafloxacin in a mouse model of cephalosporin-resistant Streptococcus pneumoniae pneumonia.

Trovafloxacin is a potentially useful agent for treatment of infections caused by cephalosporin-resistant Streptococcus pneumoniae. We studied the effectiveness of trovafloxacin therapy and examined the correlation between pharmacodynamic indices in serum and lung, and bacterial killing. Immunocompetent Balb/c mice were infected by intranasal inoculation of a cephalosporin-resistant S. pneumoniae isolate (MIC of ceftriaxone and trovafloxacin 2 and 0.06 mg/L, respectively). Trovafloxacin 10-30 mg/kg/day in one or three divided doses was started 15 h after infection. Serum and lung drug concentrations were measured at multiple time points for 24 h. Serum concentrations peaked at 30-60 min and lung concentrations approximately 30 min later. The serum T1/2 was approximately 9 h and lung T1/2 varied from 5 to 9 h. Lung AUC and Cmax values were 2-3 times greater than those in serum. At the start of therapy lung bacterial concentrations were 8.4 +/- 0.3 log10 cfu/mL and 24 h later had decreased by 3.5 +/- 0.2, 4.0 +/- 0.2, 0.8 +/- 0.3 and 1.0 +/- 1.2 log10 cfu/mL with 30 mg/kg x 1, 10 mg/kg x 3, 10 mg/kg x 1 and 3.3 mg/kg x 3 regimens, respectively. Although the larger dosages were more effective (P < 0.001) the differences between divided and single dosage regimens were not significant. Trovafloxacin serum AUC/MIC ratio correlated best with bacterial killing in the lungs over 24 h. Trovafloxacin is likely to be useful in the treatment of cephalosporin-resistant S. pneumoniae pneumonia.

Pharmacodynamics of vancomycin for the treatment of experimental penicillin- and cephalosporin-resistant pneumococcal meningitis.

With the emergence of beta-lactam antibiotic resistance among strains of Streptococcus pneumoniae, vancomycin has assumed an important role in the treatment of bacterial meningitis. Using the rabbit meningitis model, we evaluated the pharmacokinetics and pharmacodynamics of vancomycin in this setting. Animals were given 80 mg/kg of body weight daily in two or four divided doses to determine the penetration and activity of vancomycin in cerebrospinal fluid (CSF); each regimen was administered with and without dexamethasone. Mean peak (2 h) concentrations in CSF that were four- to eightfold higher than the minimum bactericidal concentration (MBC; 0.5 microgram/ml) for the pathogen were adequate for bacterial clearance. In both groups concentrations in CSF remained higher than the MBC for greater than 80% of the respective dosing intervals, and the penetration of vancomycin into CSF was 20%. Mean concentrations in CSF at 24 to 36 h of therapy were lower than those achieved during the first 12 h, consistent with a decline in the level of antibiotic entry into CSF as inflammation wanes. Rates of bacterial clearance were similar for the two regimens, and for all animals cultures of CSF were sterile by 36 h. The coadministration of dexamethasone significantly reduced the penetration of vancomycin into CSF by 29% and significantly lowered the rate of bacterial clearance during the first 6 h in animals receiving 20-mg/kg doses of vancomycin. For animals receiving 40-mg/kg doses, therapeutic peak concentrations in CSF were obtained even with steroid use, suggesting that the effect of steroids may be circumvented by the use of larger daily doses of vancomycin.