Alterations in penicillin-binding proteins of clinical and laboratory isolates of pathogenic Streptococcus pneumoniae with low levels of penicillin resistance

Several recent surveys of clinical isolates have indicated that substantial fractions of naturally occurring populations of Streptococcus pneumoniae have undergone a distinct upward move in the required minimal inhibitory concentration (MIC) of benzylpenicillin (from a range of 0.006-0.008 to 0.03-0.05 microgram/ml). Evidence is presented that in clinical pneumococcal isolates, penicillin-binding proteins (PBPs) groups 1 and 2 have a decreased affinity for radioactive benzylpenicillin as compared with penicillin-sensitive isolates from the same locale. Exposure of a penicillin-sensitive type 2 strain (MIC, 0.006 microgram/ml) to sequentially increasing concentrations of penicillin allowed the isolation of spontaneous resistant mutants with stepwise increases in the MIC of penicillin required (0.01-0.02, 0.025-0.05, and 0.1 microgram/ml), and in these laboratory isolates too, PBP groups 1 and 2 showed decreased affinity for labeled benzylpenicillin. DNA from the low-level resistant clinical or laboratory isolates could be used to transform the appropriate levels of penicillin resistance into penicillin-sensitive laboratory isolates. These findings suggest that significant fractions of natural pneumococcal populations may have acquired one or two of the low-level penicillin resistance genes.     

331株肺炎链球菌的耐药性及基因分型

目的 了解杭州地区肺炎链球菌临床株的耐药性及青霉素耐药株的分子流行病学特征。方法 用Etest法测定菌株对青霉素的最低抑菌浓度(MIC)，用纸片扩散法测定肺炎链球菌对其他8种抗生素的耐药情况。并以盒式聚合酶链反应(PCR)和青霉素结合蛋白(PBP)基因指纹等分子生物学方法分析菌株间的亲缘关系。结果临床分离得到肺炎链球菌331株，Etest法测得55株(16．6％)青霉素高度耐药株(PRSP)，127株(38．4％)青霉素中度耐药株(PISP)。纸片扩散法测得氨苄西林、复方新诺明、红霉素、四环素、利福平、氯霉素的耐药率分别为1．2％、47．7％、90％、84．3％、0．3％及13％。所有菌株对氧氟沙星、万古霉素敏感。保存存活的35株PRSP可分为17种盒式-PCR谱型，PBP2X、PBP2B、PBP1A的指纹各为5种、7种、5种。盒式谱型A、H的菌株其耐药谱、MIC值和PBP基因指纹高度一致。结论杭州地区肺炎链球菌临床株的青霉素耐药率较高，非β-内酰胺类红霉素、四环素、复方新诺明的耐药率亦较高。杭州地区可能有耐药克隆的流行。     

Horizontal transfer of penicillin-binding protein genes in penicillin-resistant clinical isolates of Streptococcus pneumoniae

Resistance to penicillin in clinical isolates of Streptococcus pneumoniae has occurred by the development of altered penicillin-binding proteins (PBPs) that have greatly decreased affinity for the antibiotic. We have investigated the origins of penicillin-resistant strains by comparing the sequences of the transpeptidase domain of PBP2B from 6 penicillin-sensitive and 14 penicillin-resistant strains. In addition we have sequenced part of the amylomaltase gene from 2 of the sensitive and 6 of the resistant strains. The sequences of the amylomaltase gene of all of the strains and of the PBP2B gene of the penicillin-sensitive strain show that S. pneumoniae is genetically very uniform. In contrast the PBP2B genes of the penicillin-resistant strains show approximately equal to 14% sequence divergence from those of the penicillin-sensitive strains and the development of penicillin resistance has involved the replacement, presumably by transformation, of the original PBP2B gene by a homologous gene from an unknown source. This genetic event has occurred on at least two occasions, involving different sources, to produce the two classes of altered PBP2B genes found in penicillin-resistant strains of S. pneumoniae. There is considerable variation among the PBP2B genes of the resistant strains that may have arisen by secondary transformation events accompanied by mismatch repair subsequent to their original introductions into S. pneumoniae.     

Penicillin-resistant viridans streptococci have obtained altered penicillin-binding protein genes from penicillin-resistant strains of Streptococcus pneumoniae

Penicillin-resistant strains of Streptococcus pneumoniae possess altered forms of penicillin-binding proteins (PBPs) with decreased affinity for penicillin. The PBP2B genes of these strains have a mosaic structure, consisting of regions that are very similar to those in penicillin-sensitive strains, alternating with regions that are highly diverged. Penicillin-resistant strains of viridans groups streptococci (e.g., S. sanguis and S. oralis) that produce altered PBPs have also been reported. The PBP2B genes of two penicillin-resistant clinical isolates of S. sanguis were identical in sequence to the mosaic class B PBP2B genes found in penicillin-resistant serotype 23 strains of S. pneumoniae. Emergence of penicillin resistance appears to have occurred by the horizontal transfer of an altered PBP2B gene from penicillin-resistant S. pneumoniae into S. sanguis. The PBP2B genes of three penicillin-resistant S. oralis strains were similar to the mosaic class B PBP2B gene of penicillin-resistant strains of S. pneumoniae but possessed an additional block of diverged sequence. Penicillin resistance in S. oralis has also probably arisen by horizontal transfer of this variant form of the class B mosaic PBP2B gene from a penicillin-resistant strain of S. pneumoniae.     

Treatment and diagnosis of infections caused by drug-resistant Streptococcus pneumoniae.

Drug-resistant strains of Streptococcus pneumoniae have now been reported from all continents and have become the predominant pathogens in some areas; many strains are resistant to multiple agents. Because of the importance of pneumococci in the etiology of meningitis, the criteria used to assess susceptibility are conservative: strains with minimal inhibitory concentrations (MICs) of penicillin G of less than or equal to 0.06 microgram/mL are regarded as susceptible, those with MICs of 0.1-1 microgram/mL are considered intermediately resistant, and those with MICs of greater than 1 microgram/mL are designated highly resistant. The diagnosis, treatment, and prevention of infections due to resistant pneumococci are the subjects of this review. Methods of susceptibility testing have now been well defined for pneumococci. Screening for penicillin resistance with 1-microgram oxacillin disks is recommended for all clinically significant isolates. The activity of other beta-lactam agents against penicillin-resistant strains has been documented, and the MICs of a number of non-beta-lactam agents have been determined as well. Treatment of resistant pneumococcal infections depends on the site of infection, the degree of resistance to penicillin G, the resistance of the infecting strain to other agents, the severity of disease, the presence of underlying conditions, and the dose and route of administration of antimicrobial agents. Current recommendations for treatment are based on retrospective case studies, and adequate prospective studies providing more definitive data are needed. Prevention of pneumococcal infections in children less than 2 years of age and in the elderly remains a problem. Improved vaccines must be developed for this purpose.     

Meta-analysis of antibiotic susceptibility and the genotype of penicillin-binding proteins in Streptococcus pneumoniae

To further understanding of the mechanisms of development of resistance to penicillin in Streptococcus pneumoniae, and the role of penicillin-binding proteins (PBPs) mutations to antibiotics resistance a meta-analysis was performed. Major databases, Pubmed, Current Contents, Biosis previews, Web of Science, were searched for studies that published within 1997 through to 2007, and reported the penicillin MIC and the alteration of PBP 1a, 2b and 2x (genes or proteins) of clinical S. pneumoniae isolates. Papers were reviewed by 2 persons and used standard criteria to enroll them. Meta-analysis was performed using a random-effects model. Overall, 20 studies were included in the meta-analysis. For the included 1771 clinical S. pneumoniae isolates, the susceptibility to penicillin decreased in inverse proportion to the presence of mutated pbp genes. The mutations of the conserved amino acid motifs STMK and SRNVP of PBP 1A, STMK and LKSG of PBP2X, and SSNT of PBP2B are critical for the penicillin resistance. Those motifs can be used as markers for the penicillin susceptibility of S. pneumoniae. These results are useful in helping define the mechanism of penicillin resistance in S. pneumoniae.     

Antimicrobial susceptibility of non-penicillinase and penicillinase-producing Neisseria gonorrhoeae strains isolated in Tripoli, Libya

Neisseria gonorrhoeae culture gave a positive result in 42 of 64 male adults with purulent urethral discharge. The majority of the infections were acquired outside Libya. Twenty-seven strains (64.3%) were non-penicillinase producing (NPPNG) and 15 (35.7%) were penicillinase producing (PPNG) by starch paper technique. Antimicrobial susceptibility of the strains to 5 antibiotics was carried out by agar-plate dilution technique. Twenty-three NPPNG strains (54.8%) were susceptible to penicillin with a minimum inhibitory concentration (MIC) of less than or equal to 0.5 micrograms/ml. In 4 strains (9.5%), a high resistance to penicillin (MIC greater than or equal to 16 micrograms/ml) appeared to be chromosomally-mediated (CMRNG). All PPNG strains were resistant to penicillin (MIC greater than or equal to 1 microgram/ml). While resistance to erythromycin (MIC greater than or equal to 1 microgram/ml) and tetracycline (MIC greater than or equal to 1 microgram/ml) was observed in 5 strains, resistance to kanamycin (MIC 32 micrograms/ml) and spectinomycin (MIC 64 micrograms/ml) was present in only one strain. Whereas no significant differences were recorded in MICs of erythromycin, tetracycline, kanamycin and spectinomycin between NPPNG and PPNG strains, one PPNG strain was found to be resistant in vitro to all 5 antibiotics.     

Susceptibility of Neisseria gonorrhoeae isolates from Jakarta, Indonesia, to antibiotics

The minimum inhibitory concentration (MIC) of penicillin, tetracycline and spectinomycin was determined for 6 beta-lactamase (PPNG) and 73 non beta-lactamase producing Neisseria gonorrhoeae isolates from Jakarta, Indonesia. All PPNG were resistant to greater than or equal to 128 micrograms/ml penicillin. Forty-six percent of the non-PPNG strains were inhibited by less than or equal to 1.0 microgram/ml of penicillin and 97% by less than or equal to 4 micrograms/ml. Most of the PPNG and non-PPNG isolates (90%) were inhibited by less than or equal to 4 micrograms/ml tetracycline and less than or equal to 16 micrograms/ml spectinomycin. Two non-PPNG strains were resistant to 64 micrograms/ml of tetracycline, 8 micrograms/ml penicillin and 32 micrograms/ml spectinomycin. The non-PPNG strains generally were more resistant to penicillin and tetracycline compared to strains tested from other parts of the world in previously reported studies.     

Antigenic variation of penicillin-binding proteins from penicillin-resistant clinical strains of Streptococcus pneumoniae.

Penicillin-resistant strains of Streptococcus pneumoniae that are isolated with increasing frequency worldwide contain low-affinity penicillin-binding proteins (PBPs). The relatedness of PBPs from 55 resistant strains isolated on three continents was investigated by testing the reactivity of antibodies specific for PBP 1a or 2b and by comparing the PBP patterns. Seventeen patterns of antibody reactivity could be distinguished, 12 of which were specific to one isolate. Most strains, including all German and South African strains, had a unique PBP profile. A few groups of Spanish and Finnish isolates were identified where the strains within each group shared the same PBP profile, the same antigenic variants of PBPs 1a and 2b, and the same serogroup, suggesting that they represent different clones of S. pneumoniae. The results demonstrated highly variable pathways of resistance development and confirmed that resistant strains have emerged independently in different locations.     

Geographic distribution of penicillin-resistant clones of Streptococcus pneumoniae: characterization by penicillin-binding protein profile, surface protein A typing, and multilocus enzyme analysis.

Examination of several hundred penicillin-resistant clinical isolates of Streptococcus pneumoniae has revealed extensive strain-to-strain variation in the number and molecular size of penicillin-binding proteins (PBPs). This polymorphism has been used to classify resistant isolates into groups (PBP families) that share distinct electrophoretic profiles. We describe herein properties of four such PBP families: two from Spain (and/or Ohio) and one each from Hungary and Alaska. We have discovered that representative isolates assigned to each PBP family also share capsular serotype, antibiotic resistance pattern, pneumococcal surface protein A type, and multilocus enzyme genotype. The results demonstrate independent clonal origin for strains assigned to each PBP family. Each resistant clone occurs with uniquely high incidence within specific geographic areas.     

Nasopharyngeal carriage of multidrug-resistant Streptococcus pneumoniae in institutionalized HIV-infected and HIV-negative children in northeastern Romania.

OBJECTIVES: The study compared nasopharyngeal carriage of resistant pneumoniae in human immunodeficiency virus (HIV)-seropositive and -seronegative children. METHODS: Nasopharyngeal colonization with Streptococcus pneumoniae was investigated during May 1996 in 162 HIV-negative infants and children (age range, 1-38 mo) and 40 HIV-infected children (age range, 39-106 mo) living in an orphanage in Iasi, northeastern Romania. The HIV-infected children lived separated from the other children and were cared for by a different staff. Streptococcus pneumoniae was isolated from 12 of 40 (30%) HIV-infected and from 81 of 160 (50%) HIV-negative children. Antimicrobial susceptibility to penicillin and ceftriaxone was determined by E-test, and to another five antibiotics by disk diffusion. Serotyping was performed by the Quellung method on 81 of 93 (87%) isolates. RESULTS: Serotypes 6A, 6B, 19A, and 23F together represented 98% of all isolates. Ninety-nine percent of S. pneumoniae isolates were resistant to penicillin, and 74% were highly resistant to penicillin (minimum inhibitory concentration [MIC] > 1 mg/mL); MIC50 and MIC90 to penicillin of the isolates were 2 mg/mL and 8 mg/mL, respectively. Eighty-nine of ninety-one isolates were susceptible to ceftriaxone; 99%, 87%, 87%, 48%, and 21% of the isolates were resistant to trimethoprim-sulphamethoxazole, erythromycin, clindamycin, tetracycline, and chloramphenicol, respectively. Eighty-two (89%) isolates were multidrug resistant (resistant to =/>3 antibiotic classes); 37 of 92 (40%) isolates were resistant to 5 or more antibiotic classes, and 16 of these 37 (43%) belonged to serotype 19A. All serotype 19 isolates were highly resistant to penicillin. CONCLUSIONS: No significant differences were observed in the resistance rates of S. pneumoniae in HIV-infected children compared to HIV-negative children. Multidrug-resistant pneumococci were highly prevalent in this Romanian orphanage in both HIV-negative and older HIV-infected children. The observed high prevalence of multidrug-resistant pneumococci (coupled with high penicillin resistance) with a limited number of circulating serotypes emphasizes the need to further evaluate the conjugate vaccines in children at risk for invasive pneumococcal infection.     

Antimicrobial resistance in Streptococcus pneumoniae: a South African perspective.

Resistance to penicillin among South African strains of Streptococcus pneumoniae increased from 4.9% in 1979 to 14.4% in 1990. Except for resistance to co-trimoxazole (44%), resistance to other antimicrobial agents remained relatively low. Multiply resistant strains belonged mainly to serovars 6B, 19A, 14, and, more recently, 23F. Use of chloramphenicol to treat meningitis caused by strains relatively resistant to penicillin proved to be unsatisfactory, probably because of the inadequate bactericidal activity of chloramphenicol against these strains. Spread of penicillin-resistant nasopharyngeal strains in pediatric wards was most common among children who received antimicrobial therapy. Penicillin-binding protein (PBP) patterns were shown to vary in resistant clinical strains. Interspecies transfer of penicillin resistance between Streptococcus mitis and S. pneumoniae was demonstrated and antigenic homology was found in PBPs 1A and 2B of strains belonging to these species. Restriction enzyme mapping following DNA amplification of the PBP 2B gene revealed six arrangements among South African strains within serogroup 19. Despite extensive studies in South Africa and several other countries, many questions with regard to the global problem of antimicrobial resistance among S. pneumoniae strains remain unanswered, especially those that relate to prevalence in developing regions of the world.     

Erythromycin-resistant group G streptococci in an isolated northern Canadian community

The susceptibility of groups A, C, and G streptococci isolated from pharynx or skin in two northern Canadian native communities during a one year study of the epidemiology of streptococcal infection was determined for penicillin, erythromycin and clindamycin using an agar dilution method. Organisms studied included 725 group A, 82 group C, and 184 group G streptococci. All organisms were susceptible to penicillin (minimum inhibitory concentration [MIC] range less than 0.004 to 0.015 μg/mL; MIC(90) 0.015 μg/mL) and clindamycin (range 0.007 to 0.06 μg/mL; MIC(90) 0.06 μg/mL) with no differences observed between streptococcal groups. For erythromycin, groups A and C were generally susceptible (range less than 0.007 to 0.030 μg/mL; MIC(90) 0.03 μg/mL; and range 0.007 to 1.0 μg/mL; MIC(90) 0.06 μg/mL, respectively). Group G was less susceptible (range 0.007 to greater than 2.0 μg/mL; MIC(90) greater than 2.0 μg/mL) with 38% of all isolates having an MIC greater than or equal to 1 μg/mL. On review of group G isolates, 100 of 100 from one community were susceptible (MIC less than 0.007 to 0.03 μg/mL) and 73 (87%) of 84 from the second community were resistant. All resistant strains tested were type T16. These data suggest that erythromycin-resistant group G streptococci may occur with high prevalence in certain populations and that patterns of antimicrobial susceptibility in isolated communities may be highly community-specific.     

Major related sets of antibiotic-resistant Pneumococci in the United States as determined by pulsed-field gel electrophoresis and pbp1a-pbp2b-pbp2x-dhf restriction profiles

To assess the genetic diversity of pneumococci causing serious disease within the United States, restriction profiles of 3 penicillin-binding protein (PBP)-gene amplicons and the dhf amplicon were examined in 241 recent sterile-site isolates from 7 population centers. This analysis provided markers useful for epidemiologic studies and was generally predictive of resistances to beta-lactam antibiotics and trimethoprim-sulfamethoxazole. Eight pulsed-field gel electrophoresis (PFGE) types, each representing 3-40 isolates, accounted for 134 of the 144 beta-lactam-resistant pneumococci (MICs >/=1 microgram/mL for penicillin, cefotaxime, or both). Five of these PFGE types contained subtypes highly related to subtypes of previously characterized pneumococcal clones. Within 4 of these PFGE types, the major composite PBP gene-dhf profile was highly related to the composite profile from the previously characterized related clone. Eight capsular serotypes were found among the 144 beta-lactam-resistant pneumococci. Divergent capsular types among isolates with identical PBP gene-dhf profiles and related PFGE types indicated several instances of capsular serotype switching.     

Characteristics and antibiotic therapy of adult meningitis due to penicillin-resistant pneumococci

Of 66 episodes of pneumococcal meningitis seen in Bellvitge Hospital, Barcelona, Spain (January 1981 to June 1987), 15 (23 percent) were due to penicillin-resistant pneumococci [minimal inhibitory concentrations (MICs) of 0.1 to 4 micrograms/ml]. Fifty percent of these strains were also resistant to chloramphenicol. Most were sporadic community-acquired cases. Clinical characteristics were similar in both penicillin-resistant and penicillin-sensitive cases. Those cases with MICs of greater than 1 microgram/ml did not show a response to penicillin therapy. Of nine patients treated with cefotaxime (200 to 350 mg/kg per day) with penicillin G MICs of 0.1 to 4 micrograms/ml and cefotaxime MICs of less than or equal to 0.03 to 1 microgram/ml, seven recovered, one experienced a relapse after 14 days of therapy and the infection was cured with intravenous vancomycin, and one patient died with sterile cerebrospinal fluid. Thus, adults with meningitis due to penicillin-resistant pneumococci may be adequately treated with high doses (around 300 mg/kg per day) of intravenous cefotaxime if MICs of penicillin G are less than or equal to 4 micrograms/ml. Cases with higher resistance may require another antibiotic such as vancomycin.     

High-level penicillin resistance among isolates of enterococci. Implications for treatment of enterococcal infections

STUDY OBJECTIVE: To determine the extent and clinical significance of high-level penicillin-resistant Enterococcus faecium at our institution. DESIGN: Surveillance of clinical enterococcal isolates, in-vitro susceptibility and timed survival studies, and determination of antibiotic efficacy in an experimental model of enterococcal endocarditis. MEASUREMENTS AND MAIN RESULTS: For a 6-month period, 14% of enterococcal isolates (30 of 212) were identified as E. faecium. One third of the isolates were highly resistant to penicillin G (minimum inhibitory concentration [MIC], greater than or equal to 200 micrograms/mL) but did not produce beta-lactamase. The findings from in-vitro survival studies showed that this high-level resistance resulted in the loss of bactericidal activity normally observed when an aminoglycoside antibiotic agent is combined with penicillin. An experimental rat model of endocarditis provided in-vivo data that confirmed our in-vitro observations. After the rats received therapy for 72 hours, penicillin G either alone or in combination with gentamicin did not significantly decrease the numbers of enterococci in vegetations on heart valves compared with untreated controls (P = 0.62 and P = 0.58, respectively). CONCLUSIONS: Enterococcus faecium accounts for a notable proportion of clinical enterococcal isolates. Many strains from patients at our institution, as well as from patients at other institutions throughout the country, are highly resistant to penicillin. Because high-level penicillin resistance has important therapeutic implications, periodic surveillance and MIC testing of significant enterococcal isolates, especially E. faecium, are suggested.     

Antimicrobial resistance in Streptococcus pneumoniae: an epidemiological survey in France, 1970-1990.

The antimicrobial resistance of Streptococcus pneumoniae was surveyed in 1970-1990 at Saint Joseph and Broussais hospitals in Paris (3,279 isolates) and in 1984-1990 at the National Reference Center for Pneumococci (NRCP) in Créteil (8,128 isolates). All isolates were tested for susceptibility and serotyped. At St. Joseph and Broussais hospitals, the rate of resistance to tetracycline increased from 14% in 1970 to 46.5% in 1978 and then decreased to approximately 20% in 1988-1990. Resistance to chloramphenicol appeared in 1972; its frequency remained at less than 10% until 1990. Resistance to macrolides was first detected in 1976, increased to 20% in 1984, and reached 29% in 1990. Among strains submitted to the NRCP, resistance to penicillin (MIC, greater than or equal to 0.1 mg/L) remained infrequent (less than or equal to 1.1%) between 1984 and 1986 but then increased steadily, reaching 12% in 1990. The frequency of high-level resistance to penicillin (MIC, greater than 1 mg/L) among penicillin-resistant pneumococci increased from 13% in 1988 to 48% in 1990. Compared with other serotypes, the penicillin-resistant serotype isolated most frequently (23F, 49.3%) was more often highly resistant to penicillin and was more often multiresistant.     

A case of bacteremia due to resistant Streptococcus pneumoniae.

Streptococcus pneumoniae isolates with varying degrees of resistance to penicillin have been described. Strains that are highly resistant to penicillin (MIC, greater than 1 microgram/mL) and that are resistant to multiple antibiotics have been reported primarily in South Africa and Spain. We report a case of an adult patient with bacteremia due to S. pneumoniae that was highly resistant to penicillin (MIC, 4 micrograms/mL) and resistant to erythromycin, clindamycin, chloramphenicol, and co-trimoxazole who was successfully treated with vancomycin.     

Mutation of penicillin-binding protein genes and antimicrobial susceptibility of Haemophilus influenzae isolated from spinal fluid or blood in children

Between September 1999 and August 2001, we studied serotypes to capsular antigen, beta-lactamase production, mutation of penicillin binding protein (PBP) genes by PCR method, and antimicrobial susceptibilities of 13 strains of Haemophilus influenzae isolated from spinal fluid or blood in children. Diseases of patients were meningitis in 11, pneumonia in 1, and laryngitis in 1. The age range of the patients was from 26 days to 5 years. The serotypes of all strains were b. Four of the 13 strains were beta-lactamase-positive. The mutation of genes of pbp3 was revealed from 4 isolates and 2 of the strains were beta-lactamase-positive. MICs of ampicillin to beta-lactamase-negative strains ranged from 0.125 to 1 microgram/ml and those to beta-lactamase-positive were more than 32 micrograms/ml. MICs of 2 strains of beta-lactamase-negative and mutation-positive were 0.5 and 1 microgram/ml. The excellent active antimicrobials in our study was cefotaxime (MIC90 0.06 microgram/ml), meropenem (MIC90 0.125 microgram/ml), ceftazidime (MIC90 0.25 microgram/ml), and cefepime (MIC90 0.25 microgram/ml).     

Genetic analyses of beta-lactamase negative ampicillin-resistant strains of Haemophilus influenzae isolated in Okinawa, Japan

Recently, the instance of beta-lactamase-negative ampicillin (AMP)-resistant (BLNAR) strains of Haemophilus influenzae has exhibited a marked increase in Japan. Our group determined the MICs of 160 clinical isolates of H. influenzae at a university hospital in Okinawa, the southernmost part of Japan, and found that 27 strains were BLNAR, while 24 strains were beta-lactamase-producing. Among the latter, 2 strains were resistant to AMP/clavulanic acid. BLNAR strains were shown to be more resistant to cephems than non-BLNAR strains. The competitive affinity assay using biotinylated AMP for penicillin-binding protein (PBP) showed that the binding of cefotiam to PBP 3A/3B of BLNAR strain C2163 was lower than that of the AMP-susceptible strain, while bindings to other PBPs were not changed. The sequences of ftsI, the gene encoding transpeptidase domain of PBP 3A and/or PBP 3B, were determined, and it was found that sequences of the ftsI gene of BLNAR strains were heterogeneous mutations. Deduced amino acid sequence analyses of BLNAR strains showed that three residues (Asn-526, Val-547, and Asn-569) were replaced with Lys, Ile, and Ser, respectively. In addition, some BLNAR strains had an additional three residues (Met-377, Ser-385, and Leu-389) in ftsI replaced with Ile, Thr, and Phe, respectively. Furthermore, changes from Asp-350 to Asn-350 and from Ser-357 to Asn-357 were also found in most BLNAR strains. These substitutions were located around the penicillin binding sites of PBP3. Multiple substitutions in the amino acid sequence seemed to be closely related with extended resistance against beta-lactams, including third-generation cephems. Randomly amplified polymorphism DNA fingerprinting of clinical isolates of BLNAR strains showed genetic heterogeneity of the strains, suggesting that the prevalence of BLNAR in this region was a result of the emergence of multiple clones of this phenotype.