Altered Penicillin-binding Protein Research Articles

Whole genome sequencing of penicillin-resistant Streptococcus pneumoniae reveals mutations in penicillin-binding proteins and in a putative iron permease

BackgroundPenicillin resistance in Streptococcus pneumoniae is mediated by a mosaic of genes encoding altered penicillin-binding proteins (PBPs). Nonetheless, S. pneumoniae has also developed non-PBP mechanisms implicated in penicillin resistance. In this study, whole genome sequencing of resistant organisms was used to discover mutations implicated in resistance to penicillin.ResultsWe sequenced two S. pneumoniae isolates selected for resistance to penicillin in vitro. The analysis of the genome assemblies revealed that six genes were mutated in both mutants. These included three pbp genes, and three non-pbp genes, including a putative iron permease, spr1178. The nonsense mutation in spr1178 always occurred in the first step of the selection process. Although the mutants had increased resistance to penicillin, the introduction of altered versions of PBPs into a penicillin-susceptible strain by sequential transformation led to strains with a minimal increase in resistance, thus implicating other genes in resistance. The introduction by transformation of the non-PBP recurrent mutations did not increase penicillin resistance, but the introduction of the nonsense mutation in the putative iron permease spr1178 led to a reduced accumulation of reactive oxygen species following exposure to penicillin and to other bactericidal antibiotics as well.ConclusionsThis study indicates that the selection of resistance to penicillin in S. pneumoniae involves the acquisition of mutations conferring tolerance to the antibiotic-induced accumulation of oxidants, which translates into an increased survival that putatively enables the selection of major resistance determinants such as mutations in PBPs.

Absence of meca gene in methicillin-resistant staphylococcus aureus isolates

Methicillin-resistant Staphylococcus aureus has emerged as a serious threat to public health, causing both hospital and community-associated infections. The gold standard for MRSA detection isthe amplification of the mecA gene that codes for the production of the altered penicillin-binding protein (PBP2a) responsible for classical methicillin resistance. This work determined the nature of methicillin-resistance observed in staphylococcal isolates. Staphylococcus aureus isolates with phenotypic resistance to methicillin (oxacillin) were tested for the carriage of the mecA gene by multiplex PCR to detect and type the SCCmec. The isolates were tested for the production of thealtered PBP2a by latex agglutination test and β-lactamase production/hyper production by microplate Nitrocefin assay. None of the isolates hybridized with any of the 16 sets of primers representing thefive major SCCmec types, nor contained the mecA gene; and none was positive for the gene product PBP2a determined by the MRSA screen latex agglutination test. Majority of the isolates 72.2 % (26/36) tested positive for β-lactamase production while 11/26 (42.3%) were β-lactamase hyper producers. The MRSA phenotype observed in the isolates was not the classical mecA-mediated resistance, but most probably due to hyper-production of β-lactamase. Reports of loss of the mecA gene (believed to be stable) during storage and the fact that all PCR detection of mecA gene reported in Nigeria were done outside the country calls for attention on building local capacity for prospectivemolecular screening for MRSA in clinical and environmental isolates to adequately document their prevalence and monitor the increase. Appropriate guidelines should also be drawn up for the properscreening and reporting of MRSA isolates with the establishment of regional Reference Laboratories.Key words: MRSA, mecA, Staphylococcus aureus, penicillin-binding protein, PBP2a.

A veterinary perspective on methicillin-resistant staphylococci

To familiarize the reader with the epidemiology, diagnosis, and infectious and zoonotic potential of methicillin-resistant staphylococci. Original research publications, scientific reviews and abstracts, case reports, and conference proceedings. Staphylococcus aureus is a common human commensal organism; acquisition of genes encoding an altered penicillin-binding protein confers resistance to beta-lactam antimicrobial drugs. Methicillin-resistant S. aureus (MRSA) are often resistant to non-beta-lactam antimicrobial drugs as well. Originally described as an important cause of nosocomial infection, MRSA colonization and infection are now often identified in humans outside healthcare settings. Like other S. aureus, MRSA may be present without clinical illness. However, when they do cause infection the consequences can be extremely serious. The major domestic animal species, including pets and livestock, may become contaminated, colonized, or infected with methicillin-resistant staphylococci, including MRSA. Dogs and cats are more likely to be colonized/infected with Staphylococcus pseudintermedius than S. aureus, but this pathogen can acquire genes encoding methicillin resistance (ie, MRSP). Diagnosis of MRSA or MRSP has implications not only for treatment of infected animals, but for potential zoonotic transmission. MRSA infection is an important cause of morbidity and mortality in humans. Animals may be contaminated, colonized, or infected with MRSA, with implications for the animal's health and as a potential reservoir for human infection. Staphylococci other than S. aureus may also acquire genes for methicillin resistance, and these species can also result in animal and occasionally human morbidity or mortality.

Prevalence and type distribution of human papillomavirus infection in women from Datong, China

This survey of 931 cervical specimens in women from Datong, China indicates that the overall human papillomavirus (HPV) prevalence was 18.6%, and the most prevalent high-risk HPV types were 16, 58, 18, 52 and 33. This study demonstrates the epidemiology of HPV infection in Datong and the potential impact of vaccination in this region.

Drug‐resistant pneumococci in children with acute lower respiratory infections in Vietnam

Acute lower respiratory infections (ALRI), primarily pneumonia, are the leading cause of death in children under 5 years of age. Most of these deaths occur in Africa and southeast Asia. Increasing rates of drug resistance in pneumococcal strains emphasize the necessity of prevention of pneumococcal vaccines. The aim of the present study was to determine the frequency of drug resistance and the distribution of serotype of pneumococcal strains isolated from pediatric patients with ALRI in Vietnam. Two hundred and twenty pediatric patients with ALRI under 5 years of age were enrolled in Hanoi, Vietnam between 2001 and 2002. Bacterial pathogens with a heavy growth (10(6) c.f.u./mL) were isolated from nasopharyngeal secretions on quantitative culture. Fifty-three pneumococcal strains isolated from the nasopharynx of pediatric patients were examined for antibiotic susceptibility including drug-resistant genes and serotyping. A total of 73.6% of pneumococcal strains were genotypic penicillin-resistant Streptococcus pnemoniae (gPRSP), possessing altered penicillin-binding protein genes pbp 1a + 2x + 2b; 67.9% of these strains were gPRSP and simultaneously had the ermB gene, which is responsible for high resistance to erythromycin. The majority of gPRSP strains were serotype 19F or 23F. gPRSP strains with serotype 19F or 23F are highly prevalent among pediatric patients with ALRI under 5 years of age in Hanoi, Vietnam.

Low β-Lactamase-Negative Ampicillin-Resistant Haemophilus influenzae Strains Are Best Detected by Testing Amoxicillin Susceptibility by the Broth Microdilution Method

Ampicillin resistance in Haemophilus influenzae due to alterations in penicillin-binding proteins (beta-lactamase negative ampicillin resistant [BLNAR]) is acquiring increasing clinical and epidemiological importance. BLNAR strains with low ampicillin MICs (0.5 to 4 microg/ml) represent the majority of this population in Europe and the United States, but separating them from susceptible isolates is challenging. To investigate the best method to identify low-BLNAR strains, we studied the antibiotic susceptibilities of 94 clinical isolates of H. influenzae by microdilution, Etest, and disk diffusion: 25 had no resistance mechanisms (gBLNAS), 34 had mutations in the ftsI gene only (gBLNAR), 20 were beta-lactamase producers only (gBLPAR), and 15 showed beta-lactamase production and mutations in the ftsI gene (gBLPACR). By current CLSI breakpoints, most gBLNAR isolates were ampicillin susceptible by microdilution (76.5%) or by Etest (88.2%). Most gBLNAR strains (79.4%) were nonsusceptible to amoxicillin (the most widely used community antibiotic in the United States and Europe) when tested by microdilution. By Etest, 15% of beta-lactamase-positive isolates were nonresistant to ampicillin or amoxicillin. The poorest agreement between Etest and microdilution results was for the gBLPAR isolates (25% for ampicillin, 15% for amoxicillin, and 10% for cefaclor). Low-strength disks of ampicillin and amoxicillin-clavulanic acid poorly identified low-BLNAR isolates and are not recommended as a screening method. We suggest new amoxicillin breakpoints for BLNAR isolates as follows: susceptible, MIC < or = 0.5 microg/ml (no resistance mechanisms; pharmacokinetic/pharmacodynamic [PK/PD] data favorable); intermediate, MICs = 1 to 2 microg/ml (resistance mechanisms present but PK/PD data favorable), and resistant, MICs > or = 4 microg/ml (resistance mechanisms present and PK/PD data unfavorable).

Pharmacokinetic and pharmacodynamic profile of ceftobiprole

A new addition to our therapeutic armamentarium against antimicrobial-resistant pathogens is ceftobiprole, a novel broad-spectrum cephalosporin currently undergoing investigation for the treatment of complicated skin and skin structure infections (cSSSIs) and nosocomial pneumonia. Several qualities make ceftobiprole uniquely suited for early empiric use. A major advance from contemporary β-lactams, ceftobiprole has a high affinity for the altered penicillin-binding protein (PBP) 2′ (2a), making it active against methicillin-resistant staphylococci. It also binds avidly to the relevant PBPs of most Gram-positive and Gram-negative pathogens, and is resistant to hydrolysis by many β-lactamases, making it uniquely suited for infections caused by Gram-positive and mixed Gram-negative organisms. This review summarizes the pharmacokinetic and pharmacodynamic profile of ceftobiprole and addresses in detail the population pharmacokinetic and Monte Carlo simulation analyses used to determine the candidate doses for the cSSSI and nosocomial pneumonia phase 3 clinical trials. This review will also address the ability of the selected dosing regimens in providing adequate free drug concentrations in excess of the MICs against a contemporary group of bacterial isolates from a global resistance surveillance study.

Antimicrobial susceptibility of Haemophilus influenzae and Moraxella catarrhalis isolates in eight Central, East and Baltic European countries in 2005-06: results of the Cefditoren Surveillance Study

Sir, Cefditoren pivoxil is an aminothiazolyl oral cephalosporin with a broad spectrum of activity against Gram-positive and Gram-negative pathogens and is stable to hydrolysis by many common b-lactamases. It has been shown to be active in vitro against many respiratory tract pathogens including Haemophilus influenzae and Moraxella catarrhalis. Resistance of both organisms to b-lactam antibiotics is generally conferred by either the presence of b-lactamase or as a result of alterations in penicillinbinding protein 3. In this study, we compare the in vitro activity of cefditoren with that of nine other antibiotics against 665 and 133 clinical isolates of H. influenzae and M. catarrhalis strains collected from eight Central, East and Baltic European countries. The isolates were prospectively collected from 1 November 2005 to 31 December 2006, in 25 centres from the Czech Republic, Hungary, Poland, Romania, Slovakia, Estonia, Latvia and Lithuania. Isolates from Estonia, Latvia and Lithuania were analysed all together as ‘Baltic countries’. No isolates of M. catarrhalis were received from Romania. Strain isolates were from patients with upper (ear and sinus exudates) and lower (sputum, bronchial washes and bronchial aspirates) respiratory tract infections and bacteraemia. Duplicate isolates from the same patient were not accepted. The identity of the isolates was confirmed by morphology of the colonies, growth on blood and chocolate agar, and API-NH (bioMerieux, Marcy l’Etoile, France). The following data were collected for each isolate: age group (adults/children), sex, status (outpatients/inpatients) and sample origin (upper respiratory tract/lower respiratory tract/blood). Susceptibility testing was carried out by the CLSI broth microdilution method, using dried commercial plates (Sensititre, Trek Diagnostic Systems Ltd, West Sussex, UK) reconstituted with Haemophilus test medium for H. influenzae and cation-adjusted Mueller–Hinton broth for M. catarrhalis. The antimicrobials tested were ampicillin, amoxicillin, amoxicillin/ clavulanic acid, cefditoren, cefixime, cefpodoxime, cefuroxime, cefotaxime, clarithromycin and levofloxacin. Breakpoint concentrations published by the CLSI (document M100-S16, 2006) to interpret MIC data qualitatively for H. influenzae were also applied to M. catarrhalis. b-Lactamase production was determined using the chromogenic cephalosporin Cefinase test (bioMerieux). The results of the susceptibility testing of H. influenzae isolates are shown in Table 1. Of the 665 isolates tested, 7 (1.1%) were intermediate and 62 (9.3%) resistant to ampicillin. Among ampicillin-resistant isolates, 95.2% were b-lactamase producers. Susceptibility to ampicillin was higher in isolates from the Czech Republic, Slovakia, Hungary, Poland and Baltic countries and lower in those from Romania, the difference being statistically significant (P 0.005) when compared with any of the other countries. Three (4.8%) ampicillin-resistant isolates were categorized as b-lactamase-negative, ampicillin-resistant (BLNAR); all of them having an MIC of 4 mg/L. No isolate was b-lactamase-positive, amoxicillin/clavulanic-resistant (BLPACR). All isolates were susceptible to amoxicillin/clavulanic acid, cefixime, cefpodoxime, cefuroxime, cefotaxime and levofloxacin. All H. influenzae isolates were inhibited by 0.06 mg/L cefditoren. More than 98% of the isolates were susceptible to clarithromycin and none was categorized as intermediate. No differences in susceptibility to the antibiotics tested were found comparing source, localization of the patient, age or gender. One hundred and twenty-seven of the 133 (95.5%) M. catarrhalis isolates produced b-lactamase, but only 94 (70.7%) were non-susceptible to ampicillin using CLSI breakpoints. Susceptibility to ampicillin was higher in isolates from Poland than from any other country, but the difference was not statistically significant. All isolates were susceptible to amoxicillin/ clavulanic acid, cefixime, cefpodoxime, cefuroxime, cefotaxime, clarithromycin and levofloxacin. All M. catarrhalis isolates were inhibited by 0.5 mg/L cefditoren and the MIC50/MIC90 values were 0.12/0.5 mg/L. No differences in susceptibility to the antibiotics tested were found comparing source, localization of the patient, age or gender. The prevalence of H. influenzae isolates producing b-lactamase in Europe varies from low (,6%) in Germany, The Netherlands and Italy to high (.20%) in the UK, Spain and France. – 4 Reports on surveillance studies in our screened countries are scarce but give the following results: 7.9% to 13% in the Czech Republic, 4.8% to 6.5% in Slovakia, 0% to 3.3% in Hungary, 6% to 24% in Poland – 5 and 3.0% in Estonia. Data on the prevalence of BLNAR H. influenzae isolates are variable but prevalence is usually low (,10%), – 4 although some reports have given figures of up to 20% in Poland. No BLPACR isolates were detected in the present study, and the prevalence of such organisms being very low. A high rate (95.5%) of b-lactamase-producing M. catarrhalis isolates was found in our study, with no significant differences Journal of Antimicrobial Chemotherapy doi:10.1093/jac/dkn083

Positive selection in penicillin-binding proteins 1a, 2b, and 2x from Streptococcus pneumoniae and its correlation with amoxicillin resistance development

The efficacy of β-lactam antibiotics in Streptococcus pneumoniae has been compromised because of the development of altered penicillin-binding proteins (PBPs), however, this has been less so for amoxicillin than for penicillin. Recently, there have been a number of important methods developed to detect molecular adaptation in protein coding genes. The purpose of this study is to employ modern molecular selection approaches to predict sites under positive selection pressure in PBPs, derived from a large international S. pneumoniae collection of amoxicillin resistant and susceptible isolates, and encompassing a comparative data set of 354 pbp1a, 335 pbp2b, and 389 pbp2x gene sequences. A correspondence discriminant analysis (CDA) of positively selected pbp sites and amoxicillin MIC (minimum inhibitory concentration) values is then used to detect sites under positive selection pressure that are important in discriminating different amoxicillin MICs. Molecular adaptation was evident throughout PBP2X, with numerous positively selected sites in both the transpeptidase (TP) and C-terminal domains, strongly correlated with discriminating amoxicillin MICs. In the case of PBP1A positive selection was present in the glycosyltransfer (GT), TP and C-terminal domains. Sites within the TP domain tended to be correlated with the discrimination of low from intermediate MICs, whereas sites within the C-terminal tail, with a discrimination of intermediate from fully resistant. Most of the positively selected sites within PBP2B were in the N-terminal domain and were not correlated with amoxicillin MICs, however, several sites taken from the literature for the TP domain were strongly associated with discriminating high from intermediate level amoxicillin resistance. Many of the positively selected sites could be directly associated with functional inferences based on the crystal structures of these proteins. Our results suggest that clinical emphasis on TP domain sequences of these proteins may result in missing information relevant to antibiotic resistance development.

Characterization of extended-spectrum -lactamase-producing isolates of Haemophilus parainfluenzae

To characterize the beta-lactam resistance mechanisms of two clinical isolates of cefotaxime-resistant Haemophilus parainfluenzae recovered from patients in South Africa. The relatedness of isolates and plasmids was assessed using PFGE and restriction enzyme analysis, respectively. Plasmid-mediated and chromosomally integrated bla(TEM) genes and ftsI genes were sequenced, and the plasmid-mediated bla(TEM-15) was used to transform a range of control organisms. The two isolates were found to be unique according to PFGE, but had an identical 3.7 kb plasmid encoding a TEM-15 beta-lactamase. Both isolates also had substitutions in penicillin binding protein 3 (PBP3) consistent with substitutions known to exist in beta-lactamase-negative ampicillin-resistant (BLNAR) strains of Haemophilus influenzae. The cefotaxime MICs for control strains of H. influenzae, H. parainfluenzae and BLNAR H. influenzae transformed with the plasmid-mediated bla(TEM-15) were 1.0, 1.0 and 4.0 mg/L, respectively, compared with 16.0 and 8.0 mg/L, respectively, for the two parent H. parainfluenzae. The high-level cefotaxime resistance in the H. parainfluenzae isolates was due to a combination of a plasmid-mediated TEM-15 extended-spectrum beta-lactamase with altered PBP3 probably contributing. Other contributing resistance mechanisms could not be excluded.

Heteroresistance to penicillin in Streptococcus pneumoniae

Heteroresistance to beta-lactam antibiotics has been mainly described for staphylococci, for which it complicates diagnostic procedures and therapeutic success. This study investigated whether heteroresistance to penicillin exists in Streptococcus pneumoniae. Population analysis profile (PAP) showed the presence of subpopulations with higher penicillin resistance in four of nine clinical pneumococcal strains obtained from a local surveillance program (representing the multiresistant clones ST179, ST276, and ST344) and in seven of 16 reference strains (representing the international clones Spain(23F)-1, Spain(9V)-3, Spain(14)-5, Hungary(19A)-6, South Africa(19A)-13, Taiwan(23F)-15, and Finland(6B)-12). Heteroresistant strains had penicillin minimal inhibitory concentrations (MICs) (for the majority of cells) in the intermediate- to high-level range (0.19-2.0 mug/ml). PAP curves suggested the presence of subpopulations also for the highly penicillin-resistant strains Taiwan(19F)-14, Poland(23F)-16, CSR(19A)-11, and CSR(14)-10. PAP of bacterial subpopulations with higher penicillin resistance showed a shift toward higher penicillin-resistance levels, which reverted upon multiple passages on antibiotic-free media. Convergence to a homotypic resistance phenotype did not occur. Comparison of two strains of clone ST179 showed a correlation between the heteroresistant phenotype and a higher-penicillin MIC and a greater number of altered penicillin-binding proteins (PBP1a, -2b, and -2x), respectively. Therefore, heteroresistance to penicillin occurs in international multiresistant clones of S. pneumoniae. Pneumococci may use heteroresistance to penicillin as a tool during their evolution to high penicillin resistance, because it gives the bacteria an opportunity to explore growth in the presence of antibiotics before acquisition of resistance genes.

Antimicrobial Resistance inHaemophilus influenzae

Haemophilus influenzae is a major community-acquired pathogen causing significant morbidity and mortality worldwide. Meningitis and bacteremia due to type b strains occur in areas where the protein-conjugated type b vaccine is not in use, whereas nontypeable strains are major causes of otitis media, sinusitis, acute exacerbations of chronic bronchitis, and pneumonia. Antibiotic resistance in this organism is more diverse and widespread than is commonly appreciated. Intrinsic efflux resistance mechanisms limit the activity of the macrolides, azalides, and ketolides. beta-Lactamase production is highly prevalent worldwide and is associated with resistance to ampicillin and amoxicillin. Strains with alterations in penicillin binding proteins, particularly PBP3 (beta-lactamase negative ampicillin resistant and beta-lactamase positive amoxicillin-clavulanate resistant), are increasing in prevalence, particularly in Japan, with increasing resistance to ampicillin, amoxicillin, amoxicillin-clavulanate, and many cephalosporins, limiting the efficacy of expanded-spectrum cephalosporins against meningitis and of many oral cephalosporins against other diseases. Most strains remain susceptible to the carbapenems, which are not affected by penicillin binding protein changes, and the quinolones. The activity of many oral agents is limited by pharmacokinetics achieved with administration by this route, and the susceptibility of isolates based on pharmacokinetic and pharmacodynamic parameters is reviewed.

Resistance patterns of selected respiratory tract pathogens in Poland

This study presents the results of a survey of the in-vitro susceptibility to antimicrobial agents of major pathogens responsible for community-acquired respiratory tract infections in Poland during 2002-2004. The collection of 1184 bacterial isolates comprised 398 Streptococcus pneumoniae, 344 Haemophilus influenzae, 302 Streptococcus pyogenes and 140 Moraxella catarrhalis. Among the pneumococcal isolates, 16.8% were penicillin-non-susceptible (PNSP), of which 80.6% were identified as multidrug-resistant. Overall, 9.0% of H. influenzae isolates were beta-lactamase-positive, although this percentage increased noticeably in the third year of the study. Based on PCR results, 12.8% of H. influenzae isolates were identified as low-level beta-lactamase-negative, ampicillin-resistant (BLNAR), and one isolate as low-level beta-lactamase-positive, amoxycillin-clavulanic acid-resistant (BLPACR). Pulsed-field gel electrophoresis (PFGE) classified 45 H. influenzae isolates with altered penicillin-binding proteins into 15 PFGE types, including two predominant types (with four and six sub-types) containing 15 and ten isolates, respectively. Resistance to tetracycline, erythromycin and clindamycin was found in 20.9%, 8.9% and 4.6% of S. pyogenes isolates, respectively. The production of beta-lactamase characterised 91.4% of M. catarrhalis isolates. In summary, the overall occurrence of PNSP in Poland remains stable, although there was a noticeable increase in the proportion of fully-resistant isolates. A rising trend in the prevalence of beta-lactamase producers and low-level BLNAR isolates was observed among Polish isolates of H. influenzae.

Multilaboratory Evaluation of Disk Diffusion Antimicrobial Susceptibility Testing of Neisseria meningitidis Isolates

In 2005, the Clinical and Laboratory Standards Institute published MIC interpretive criteria for 13 antimicrobial agents used for either therapy or prophylaxis of Neisseria meningitidis infections. The MIC method includes the use of lysed horse blood-supplemented Mueller-Hinton broth with incubation in 5% CO2 for 20 to 24 h. Since some clinical laboratories might prefer the option of disk diffusion testing for infrequently encountered isolates a multicenter collaborative study was conducted to evaluate the reproducibility of a disk diffusion method for testing isolates of N. meningitidis. Interpretive criteria were developed for 12 antimicrobial agents. Four laboratories tested a common collection of 50 meningococcal strains and then tested 25 unique isolates per laboratory. Isolates were tested using Mueller-Hinton sheep blood agar plates incubated for 20 to 24 h in 5% CO2; they were also tested by the reference broth microdilution method in parallel. Pooling of the MIC and disk diffusion data from the common and unique isolates provided a sufficient sample size to develop susceptible, intermediate, and resistant zone diameter interpretive criteria using the error rate-bounded method for the following agents: chloramphenicol, trimethoprim-sulfamethoxazole, ciprofloxacin, and rifampin. Due to the lack of resistant strains at the present time, "susceptible only" interpretive criteria were proposed for cefotaxime, ceftriaxone, meropenem, azithromycin, and minocycline. The numbers of minor interpretive errors with penicillin and ampicillin disk tests were unacceptably high and precluded recommended testing of those agents by the disk method. However, amdinocillin, an agent that preferentially binds to the altered penicillin binding protein responsible for diminished penicillin susceptibility, has potential utility as a surrogate screening reagent for ampicillin resistance. A disk diffusion breakpoint was derived for nalidixic acid to serve as a surrogate marker for gyrase A mutations associated with diminished fluoroquinolone susceptibility. Disk diffusion testing with meningococci can be performed in a reproducible manner with several antimicrobial agents and represents a practical and cost-effective option for testing sporadic clinical isolates or for surveillance purposes by resource-limited laboratories.

Investigation of aminoglycoside modifying enzyme genes in methicillin-resistant staphylococci

Methicillin-resistant staphylococci may also be resistant to some other antibiotics as well as beta-lactams. In this study, co-existence of resistance to methicillin and aminoglycosides was genetically investigated in staphylococci. A total of 50 staphylococci from in-patients, 17 Staphylococcus aureus and 33 coagulase negative staphylococci (CNS) that contained mecA (gene encoding PBP 2a, an altered penicillin-binding protein) determined by polymerase chain reaction (PCR) were included in the study. Aminoglycoside modifying enzyme (AME) genes were investigated using multiplex-PCR. Aminocyclitol-6'-acetyltransferase-aminocyclitol-2''-phosphotransferase [aac(6')/aph(2'')] gene (encoding bifunctional acetyltransferases/phosphotransferases) was determined in 66% of the isolates, aminocyclitol-4'-adenylytransferase (ant(4')-Ia) gene (encoding phosphotransferases) in 24%, and aminocyclitol-3'-phosphotransferase (aph(3')-IIIa) gene (encoding nucleotidyltransferases) in 8%. Two isolates contained all these three genes. Thirty-six (72%) isolates had at least one of these genes. Three CNS and one S. aureus isolates sensitive to oxacillin had the mecA gene. In conclusion, a high rate of aminoglycoside resistance was determined in methicillin-resistant staphylococci. The aac(6')/aph(2'') was the most frequently detected.

Presence of a mutation in ponA1 of Neisseria gonorrhoeae in numerous clinical samples resistant to various β-lactams and other, structurally unrelated, antimicrobials

The number of resistant strains in patients with Neisseria gonorrhoeae urethritis has been increasing, making effective treatment difficult. Chromosomally mediated penicillin-resistant N. gonorrhoeae arise through alterations in penicillin-binding proteins (PBPs) and a decrease in outer membrane permeability. To understand the occurrence of penicillin resistance in patients with N. gonorrhoeae infection, we performed this study. In addition, we studied minimum inhibitory concentrations (MICs) of antimicrobials against N. gonorrhoeae strains. We measured the MICs of penicillin G, other beta-lactams, and other kinds of antimicrobials against 53 clinical N. gonorrhoeae isolates from male patients with urethritis in Hyogo and Osaka, Japan. The ponA genes, encoding PBP 1 of these isolates, were sequenced. Of the 53 isolates tested, 41 strains showed some resistance to penicillin G. A mutation in the ponA (ponA1) gene was identified in 46 isolates. There was a tendency that ponA mutant (ponA1) in N. gonorrhoeae led to higher antimicrobial MICs of beta-lactam antimicrobial agents (including penicillin) than those of non-ponA mutants. However, we found lower than expected MICs of penicillin and beta-lactams even in ponA mutants. Therefore, we consider that detailed investigations for the further understanding of the effect of other genes, such as penC (which is reported to be related to ponA1 in achieving high-level penicillin resistance) should be our next step.

In vitro activity of ertapenem: review of recent studies.

Ertapenem is a long-acting, 1beta-methyl parenteral Group 1 carbapenem antibiotic that has a broad antibacterial spectrum and once-a-day dosing supported by clinical studies. Ertapenem is active against both Gram-positive and Gram-negative bacteria, including Enterobacteriaceae, Streptococcus pneumoniae and most species of anaerobic bacteria. Isolates from a variety of infections (intra-abdominal infections, skin/soft-tissue infections, community-acquired pneumonia, pelvic infections and urinary tract infections) are inhibited by ertapenem. It has restricted activity against nosocomial pathogens such as Pseudomonas aeruginosa, Acinetobacter species, methicillin-resistant staphylococci and enterococci. Ertapenem has potent activity against the majority of anaerobic isolates from intra-abdominal infections, and against most of the aerobes isolated from these infections, with the exceptions of the nosocomial pathogens mentioned above. MIC(90)s for most species of Enterobacteriaceae were <1 mg/L, significantly lower than those of imipenem. MIC(90)s for most Bacteroides fragilis group isolates ranged from 1 to 4 mg/L, and MIC(90)s were species specific for Clostridium, ranging from 0.06 mg/L for Clostridium perfringens to 4 mg/L for Clostridium clostridioforme. Ertapenem was equivalent to or better than piperacillin-tazobactam in activity against most anaerobic species isolated from these infections, and was more potent than piperacillin-tazobactam and ceftriaxone against the most common skin pathogens (e.g. methicillin-susceptible Staphylococcus aureus). Ertapenem was highly active against most of the pathogens isolated from patients with community-acquired pneumonia, except for isolates of methicillin-resistant S. aureus (which are infrequent causes of community-acquired infection); these isolates were also resistant to ceftriaxone. Resistance to ertapenem is most commonly attributable to a variety of mechanisms including alterations in penicillin-binding proteins in Gram-positive organisms, and combinations of potent metallo-beta-lactamase enzymes, porin protein defects and efflux pumps in Gram-negative organisms.

Contribution of beta-lactamase and PBP amino acid substitutions to amoxicillin/clavulanate resistance in beta-lactamase-positive, amoxicillin/clavulanate-resistant Haemophilus influenzae.

The roles of beta-lactamase and alterations in penicillin-binding protein in the development of amoxicillin and amoxicillin/clavulanate resistance in two beta-lactamase-positive, amoxicillin/clavulanate-resistant (BLPACR) strains of Haemophilus influenzae were investigated. Seven beta-lactamase-negative, ampicillin-resistant (BLNAR) strains were also studied for comparison of their resistance mechanisms. All strains had been recovered from patients in Japan. The TEM type beta-lactamase of the two BLPACR strains had 100% homology with the amino acid sequences of published TEM-1 beta-lactamase, showing that amoxicillin/clavulanate resistance was not associated with mutations in this beta-lactamase. However, these strains, as well as the seven BLNAR strains, had multiple mutations in ftsI, which encodes penicillin binding protein 3 (PBP3). The transformation of H. influenzae Rd strain with amplified ftsI genes from two BLPACR and two BLNAR strains enabled the selection of amoxicillin/clavulanate-resistant transformants with the same mutations as their parent strains. We concluded that amoxicillin/clavulanate resistance in the two BLPACR strains was due to changes in PBP3. The possibility of the presence of an extended spectrum beta-lactamase was excluded in the BLPACR strains studied.

Activity of oral β-lactam antimicrobial agents versus respiratory tract isolates of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in the era of antibiotic resistance

The management of outpatient respiratory tract infections with oral β-lactam antimicrobial agents has been complicated by the emergence of β-lactamase-producing strains of Haemophilus influenzae and Moraxella catarrhalis and isolates of Streptococcus pneumoniae that express varying levels of β-lactam resistance because of altered penicillin-binding proteins. There is no question that all 3 of these important respiratory tract pathogens have changed conspicuously in the context of decreased activity of β-lactam antimicrobial agents. The question arises, what does this change mean from a clinical perspective? In this review, the in vitro activity of various oral β-lactam antimicrobial agents versus contemporary isolates of H influenzae, M catarrhalis, and S pneumoniae will be compared. The approach that is used in defining organisms as being susceptible or resistant to β-lactam antimicrobial agents will be elucidated. Finally, an effort will be made to assess the ramifications of β-lactam resistance for therapeutic efficacy in patients with respiratory tract infections caused by these 3 bacteria. We conclude that, notwithstanding diminished β-lactam activity for many clinical isolates of H influenzae, M catarrhalis, and S pneumoniae, certain agents remain effective in treating outpatient respiratory tract infections. (Otolaryngol Head Neck Surg 2002;127:S17-S23.)

Effects of amino acid alterations in penicillin-binding proteins (PBPs) 1a, 2b, and 2x on PBP affinities of penicillin, ampicillin, amoxicillin, cefditoren, cefuroxime, cefprozil, and cefaclor in 18 clinical isolates of penicillin-susceptible, -intermediate, and -resistant pneumococci.

Amino acid alterations in or flanking conserved motifs making up the active binding sites of penicillin-binding proteins (PBPs) 1a, 2b, and 2x of pneumococci were correlated with changes in affinities of penicillin, ampicillin, amoxicillin, cefditoren, cefuroxime, cefprozil, and cefaclor for these PBPs. Four penicillin-susceptible (PSSP), eight penicillin-intermediate (PISP), and six penicillin-resistant (PRSP) pneumococci were studied by DNA sequencing of the penicillin-binding sites of the pbp1a, -2x, and -2b genes of strains and by determining 50% inhibitory concentrations of the seven agents for PBP1a, -2x, and -2b. Two PSSP strains had alterations in PBP2x (L(546)-->V) (one strain) or PBP2b (T(445)-->A) (one strain). All eight PISP strains had at least two alterations--T(338)-->P or A or H(394)-->Y in PBP2X and T(445)-->A in BPB2b. All PRSP strains had the same changes seen in PISP strains, as well as T(371)-->A or S substitutions in PBP1a. The two most resistant PRSP strains had a second change in PBP2x (M(339)-->F) in a conserved motif. The affinities of penicillin and ampicillin for all three PBPs were decreased for PRSP and most PISP strains. The affinity of amoxicillin for PBP1a and -2x was decreased only for PRSP. Cefaclor and cefprozil showed decreased affinity of PRSP but not PISP for all three PBPs. Cefuroxime showed decreased affinity of PISP and PRSP for PBP1a and -2x but no change for PBP2b. Cefditoren showed no difference in PBP affinity based on penicillin or cefditoren MICs, indicating a different PBP target for this agent. Overall, the MICs for and PBP affinities of the strains correlated with the changes found in the PBP active binding sites.