Amoxicillin-resistant Isolates Research Articles

Multiple amino acid substitutions in penicillin-binding protein-1A confer amoxicillin resistance in refractory Helicobacter pylori infection.

Amoxicillin resistance in Helicobacter pylori is mainly associated with mutations in penicillin-binding protein-1A (PBP-1A). However, the specific amino acid substitutions in PBP-1A that confer amoxicillin resistance in H. pylori remain to be investigated. This study aimed to investigate the molecular mechanism underlying amoxicillin resistance in patients with refractory H. pylori infection. Esophagogastroduodenoscopy (EGD) was performed in patients with persistent H. pylori infection after at least two courses of H. pylori eradication therapy between January-2018 to March-2021. Refractory H. pylori was cultured from the gastric biopsy specimens. Antibiotic susceptibility testing was conducted to determine the minimum inhibitory concentrations (MICs). Sequence analysis of pbp-1A was performed for amoxicillin-resistant strains. Thirty-nine successfully cultured isolates were classified as refractory H. pylori isolates, and seventeen isolates were resistant to amoxicillin (MIC > 0.125mg/L). Sequence analysis of resistant strains showed multiple mutations in the C-terminal region of PBP-1A that conferred amoxicillin resistance in H. pylori. However, the number of PBP-1A mutations did not correlate with the high MICs of amoxicillin-resistant isolates. Notably, some amino acid substitutions were identified in all Taiwanese isolates with history of eradication failure but not in published amoxicillin-susceptible strains, suggesting that the mutations may play a role in conferring antibiotic resistance to these strains. Our results show that amoxicillin resistance in refractory H. pylori is highly correlated with numerous PBP-1A mutations that are strain specific. Continuous improvements in diagnostic tools, particularly molecular analysis approaches, can help to optimize current antimicrobial regimens.

RESISTANCE OF KLEBSIELLA PNEUMONIAE TO ANTIBIOTICS OF CAT TREATED AT ANIMAL CLINIC

The antibiotic resistance of Klebsiella pneumoniae has become a global health concern, leading in a reduction in the efficacy of various medications. This study seeks to determine the antibiotic resistance of K. pneumoniae isolated from cats hospitalized in Depok veterinary clinics. Surveys, sample collection, isolation, identification, and antibiotic susceptibility testing comprised the research technique. Seven K. pneumoniae isolates were discovered. There were seven cefotaxime resistant isolates, three amoxicillin resistant isolates, one gentamicin resistant isolate, and one enrofloxacin resistant strain. At the same concentration, one isolate shown intermediate susceptibility to amoxicillin, enrofloxacin, and doxycycline. Sensitivity was demonstrated by six gentamicin isolates, six doxycycline isolates, five enrofloxacin isolates, and three amoxicillin isolates. This information should help veterinarians choose the best efficient antibiotic to treat infections caused by these bacteria.

Point mutations in the glycosyltransferase domain of the pbp1a gene in amoxicillin-resistant Helicobacter pylori isolates

IntroductionHelicobacter pylori (H. pylori) eradication treatment includes a proton pump inhibitor and two antibiotics: amoxicillin and clarithromycin. The goal of that treatment is to eradicate the infection in at least 90% of the patients. Failure to eradicate the infection can have multiple causes, among which is the presence of point mutations in the antimicrobial target genes. ObjectiveTo characterize the mutations present in the pbp1a gene and their possible association with resistance to amoxicillin in vitro. MethodologySusceptibility to amoxicillin was evaluated in 147 isolates of H. pylori from the Colombian municipality of Túquerres. PCR amplification and sequencing of the glycosyltransferase domain of the pbp1a gene were carried out on Túquerres isolates, and the association between mutations and resistance was evaluated. ResultsA total of 5.4% (8/147) Túquerres isolates were resistant to amoxicillin in vitro. PCR amplification of the glycosyltransferase domain of the pbp1A gene was performed on 87.5% of the amoxicillin-resistant isolates in vitro, and in the DNA sequencing analysis, a total of 2 changes of amino acids from 3 DNA mutations that encoded the PBP1A-1 protein were observed. ConclusionThe present study is the first report on pbp1a gene mutations in H. pylori isolates coming from a population in Túquerres. Mutations that have not been reported in previous studies were found.

Mutaciones puntuales en el dominio transglicosilasa del gen pbp1a en aislados de Helicobacter pylori resistentes a amoxicilina

IntroductionHelicobacter pylori (H. pylori) eradication treatment includes a proton pump inhibitor and two antibiotics: amoxicillin and clarithromycin. The goal of that treatment is to eradicate the infection in at least 90% of the patients. Failure to eradicate the infection can have multiple causes, among which is the presence of point mutations in the antimicrobial target genes. ObjectiveTo characterize the mutations present in the pbp1a gene and their possible association with resistance to amoxicillin in vitro. MethodologySusceptibility to amoxicillin was evaluated in 147 isolates of H. pylori from the Colombian municipality of Túquerres. PCR amplification and sequencing of the glycosyltransferase domain of the pbp1a gene were carried out on Túquerres isolates, and the association between mutations and resistance was evaluated. ResultsA total of 5.4% (8/147) Túquerres isolates were resistant to amoxicillin in vitro. PCR amplification of the glycosyltransferase domain of the pbp1A gene was performed on 87.5% of the amoxicillin-resistant isolates in vitro, and in the DNA sequencing analysis, a total of 2 changes of amino acids from 3 DNA mutations that encoded the PBP1A-1 protein were observed. ConclusionThe present study is the first report on pbp1a gene mutations in H. pylori isolates coming from a population in Túquerres. Mutations that have not been reported in previous studies were found.

Antibiotic resistance profile of local thermophilic Bacillus licheniformis isolated from Maysan province soil

The key concern for public health is that bacterial strains isolated from various ecosystems are immune to antibiotics used in human medicine, thus dramatically limiting therapeutic options and threatening the lives of infected people. The present study aims to reveal the antibiotics profile of fiftysix isolates of local thermophilic Bacillus licheniformis isolated from different environmental soil sites in Maysan city, Iraq. The antimicrobial agent resistance profile of B. licheniformis isolates was performed using the disc diffusion assay according to Kirby-Bauer susceptibility test protocol. The results showed that isolates were resistance against cefepime (n=56; 100%), amoxicillin (n=13; 23.3%) and ampicillin (n=52; 92.9%); and intermediate (n=56; 100%) against cephalothin and naldixic acid. The percentage resistance was low for aztreonam (n=4; 7%), chloramphenicol (n=3; 5%), clotrimazole (n=6; 10%), novobiocin (n=2; 3.5%) and ticarcillin (n=3; 5%). On the other hand, all isolates were sensitive (n=56; 100%) towards the following antibiotics: amikacin, ceftazidime, ciprofloxacin, clindamycin, imipenem, netilmycin, gentamicin, nitrofurantion, rifampin, trimethoprim and vancomycin. The results of this study suggest that the Iraqi thermophilic B. licheniformis isolates are variable in their susceptibility towards the standards antimicrobial agents. Furthermore, the presence of cefepime, amoxicillin, ampicillin, cephalothin and naldixic resistant isolates of B. licheniformis in Iraqi soils is of concern about how resistance could spread to other bacteria, and ultimately to humans.

Genome analysis of enterobacteriaceae with non-wild type susceptibility to third-generation cephalosporins recovered from diseased dogs and cats in Europe

BackgroundExtended-spectrum-β-lactamases (ESBL) and plasmid-mediated cephalosporinases (pAmpC)-producing Enterobacteriaceae isolates are now reported worldwide in humans, animals, and in the environment. We identified the determinants of resistance to β-lactams and associated resistance genes as well as phylogenetic diversity of 53 ESBL- or pAmpC-producing Enterobacteriaceae isolated from dogs and cats in Europe. Materials/methodsOf a collection of 842 Enterobacteriaceae isolates that were recovered in 2013 and 2014 from 842 diseased and untreated dogs and cats, for 242 ampicillin or amoxicillin resistant isolates (MIC ≥ 16 mg/L), cefotaxime (CTX) and ceftazidime (CAZ) MICs were determined. Isolates with CTX and/or CAZ MIC ≥ 1 mg/L (n = 63) were selected, and their genomes were fully sequenced using Illumina Technology. Genomic data were explored to identify the resistance determinants, the plasmid incompatibility groups, and the sequence types (STs). Plasmid location of blaESBL and blaAmpC was evaluated for all isolates based on the co-localization of resistance and plasmid incompatibility group genes on the same contig. Phylogenetic trees were constructed using core-genome MLST. ResultsOf the 63 sequenced isolates, 53 isolates harbored a blaESBL or blaAmpC gene. Ten CTX and/or CAZ non-wild type isolates had neither blaESBL nor blaAmpC. Among the 63 isolates, 44 (69.8 %) were Escherichia coli, 11 (17.5 %) were Klebsiella pneumoniae, and 8 (12.7 %) were Proteus mirabilis. Fifty-one (80.9 %) isolates originated from dogs and 12 (19.1 %) from cats. Isolates were sampled from urinary tract (n = 36), skin and soft tissue (n = 22) and respiratory tract infections (n = 5). Thirty-two isolates (32/53, 60.4 %) carried blaESBL genes, including blaCTX-M-15 (n = 12), blaCTX-M-14 (n = 6), blaCTX-M-1 (n = 5), blaCTX-M-2 (n = 3), blaCTX-M-27 (n = 3), blaSHV-28 (n = 4), blaSHV-12 (n = 2), and blaVEB-6 (n = 1). Four isolates of K. pneumoniae had both blaCTX-M-15 and blaSHV-28. Twenty-one isolates (21/53, 39.6 %) carried genes encoding pAmpC, including blaCMY-2 (n = 19) and blaDHA-1 (n = 2). Thirteen E. coli isolates harbored both blaESBL or blaAmpC genes and plasmids of incompatibility groups IncIB (9/13), IncI1 (8/13), and IncFII (6/13). In addition to the reduced susceptibility to CTX and/or CAZ, reduced susceptibility or evidence of acquired resistance to at least one other relevant class of antibiotics was observed for all 63 isolates. E. coliisolates clustered in 23 STs, including B2 virulent clones from humans such as ST131 (n = 5), K. pneumoniae isolates mostly clustered in 3 STs: ST11 (n = 4), ST307 (n = 3), and ST16 (n = 2). Phylogenetic analysis identified the spread of E. coli ST131 blaCTX-M-27, and of K. pneumoniae ST307 harboring blaCTX-M-15 and blaSHV-28 or ST11 blaCTX-M-15. ConclusionsWe report here a 6.3 % prevalence of ESBL/pAmpC producing Enterobacteriaceae in diseased dogs and cats. This EU survey confirms that dogs and cats can be infected with epidemic multidrug resistant clones that may also spread in humans.

Antibiotic susceptibility and molecular characterization of resistance genes among Escherichia coli and among Salmonella subsp. in chicken food chains

ObjectiveTo investigate the occurrence of resistance genes among Escherichia coli (E. coli) and Salmonella subsp. isolated in chicken food chains in Phnom Penh, 2012–2013. MethodsSix hundred eighty two E. coli and 181 Salmonella Albany, Corvallis, and Kentucky strains were examined for susceptibilities to eight antimicrobials and following resistance genes were identified by PCR: blaTem, StrA, aadA, sul1, sul2, gyrA, Tet (A), and Tet (B). ResultsE. coli presented high resistances to tetracycline, amoxicillin, and sulfamethoxazole (63.1%–76.1%). Salmonella Albany and Salmonella Kentucky traduced high resistance percentages to amoxicillin, tetracycline, sulfamethoxazole, and nalidixic acid (84.6%–100%). Among amoxicillin-resistant isolates, blaTem genes were observed for 62% of E. coli isolates and 20% of 65 Salmonella Kentucky. The StrA gene was prevalent in 36% of 331 aminoglycoside-resistant E. coli and 90% of 40 aminoglycoside-resistant Salmonella Corvallis. The sul2 gene was predominant among sulfamethoxazole-resistant isolates, for 56% of 431 E. coli and 53% of 66 Salmonella Corvallis; the sul1 gene was observed in 54% of Salmonella Albany. The Tet (A) resistance gene was prevalent in E. coli (86%), Salmonella Corvallis (82%), Salmonella Kentucky (84%). High percentages of gyrA genes observed among nalidixic-acid resistant E. coli (91%), Salmonella Albany (92%), Salmonella Corvallis (75%) and Salmonella Kentucky (85%). ConclusionsImportant occurrences of resistance gene were observed among E. coli and Salmonella in chicken food chains in Cambodia.

Antibiotic Resistance, Virulence, and Genetic Background of Community-Acquired Uropathogenic Escherichia coli from Algeria.

The aim of the study was to investigate antibiotic resistance mechanisms, virulence traits, and genetic background of 150 nonrepetitive community-acquired uropathogenic Escherichia coli (CA-UPEC) from Algeria. A rate of 46.7% of isolates was multidrug resistant. bla genes detected were blaTEM (96.8% of amoxicillin-resistant isolates), blaCTX-M-15 (4%), overexpressed blaAmpC (4%), blaSHV-2a, blaTEM-4, blaTEM-31, and blaTEM-35 (0.7%). All tetracycline-resistant isolates (51.3%) had tetA and/or tetB genes. Sulfonamides and trimethoprim resistance genes were sul2 (60.8%), sul1 (45.9%), sul3 (6.7%), dfrA14 (25.4%), dfrA1 (18.2%), dfrA12 (16.3%), and dfrA25 (5.4%). High-level fluoroquinolone resistance (22.7%) was mediated by mutations in gyrA (S83L-D87N) and parC (S80I-E84G/V or S80I) genes. qnrB5, qnrS1, and aac(6')-Ib-cr were rare (5.3%). Class 1 and/or class 2 integrons were detected (40.7%). Isolates belonged to phylogroups B2+D (50%), A+B1 (36%), and F+C+Clade I (13%). Most of D (72.2%) and 38.6% of B2 isolates were multidrug resistant; they belong to 14 different sequence types, including international successful ST131, ST73, and ST69, reported for the first time in the community in Algeria and new ST4494 and ST4529 described in this study. Besides multidrug resistance, B2 and D isolates possessed virulence factors of colonization, invasion, and long-term persistence. The study highlighted multidrug-resistant CA-UPEC with high virulence traits and an epidemic genetic background.

Bacterial etiology of acute otitis media and characterization of pneumococcal serotypes and genotypes among children in Moscow, Russia.

We aimed to describe bacterial etiology of acute otitis media (AOM) and characterize resistance, serotypes and genotype profiles of AOM-causing pneumococci recovered in Moscow children. Children with AOM and an available middle ear fluid specimen were prospectively enrolled in this study. Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenzae and Moraxella catarrhalis were considered as true otopathogens. All pneumococcal isolates were serotyped using the Quellung reaction; multidrug-resistant (MDR) pneumococci underwent multilocus sequence typing. In 172 of 541 enrolled AOM patients (32%) at least 1 otopathogen was recovered, with S. pneumoniae having the highest rate of 63% (109/172). When adjusted for antibiotic treatment before sampling, in untreated patients the rate of culture-positive AOM was 35% (124/352), S. pneumoniae had a prevalence of 69% (86/124), S. pyogenes 19% (24/124), H. influenzae 13% (16/124) and M. catarrhalis 9% (11/124). Among 107 examined pneumococci, 45% were penicillin-nonsusceptible, 34 and 30% were resistant to erythromycin and clindamycin, respectively; 30% had an MDR phenotype, but no amoxicillin-resistant isolates were found. Ten of 32 (31%) MDR pneumococci related to clonal complex 320, the remaining isolates belonged to 7 different clonal complex. Six leading serotypes were 19F (27%), 3 (12%), 6B (11%), 14 (11%), 19A (9%) and 23F (8%); overall polysaccharide conjugate vaccine13 coverage was 93%. S. pneumoniae, the leading bacterial AOM pathogen in Moscow children, is characterized by a substantial rate of antibiotic nonsusceptibility and clonality. A polysaccharide conjugate vaccine with expanded coverage seems to fit the current AOM pneumococcal serotype distribution in Russia better.

β-Lactamases, transferable quinolone resistance determinants, and class 1 integron-mediated antimicrobial resistance in human clinical Salmonella enterica isolates of non-Typhimurium serotypes

This work investigates the occurrence and features of class 1 integrons and the presence of transferable quinolone resistance determinants (TQRD) among 382 clinical Salmonella enterica isolates of non-Typhimurium serotypes as well as the β-lactamases produced by amoxicillin-resistant isolates. These isolates were recovered in 2001 and from 2004 to 2009 from patients from the health region of Terres de l’Ebre (Catalonia, Spain) and comprised 41 different serotypes, mostly of serovar Enteritidis (n=272), being 16.5% multidrug-resistant (MDR). Among the 93 amoxicillin-resistant isolates, 84 produced TEM-1,4 produced an extended-spectrum β-lactamase (CTX-M-9 in one S. Grumpensis and in one S. Virchow, CTX-M-15 in S. Kapemba, and SHV-12 in S. Enteritidis), one produced DHA-1 (S. Newport), and 4 did not present any of the investigated β-lactamases. TQRD were found in 2 isolates (qnrA1 in CTX-M-9-producing S. Grumpensis and qnrB4 in DHA-1-producing S. Newport). Overall, 35 isolates (9.2% of all isolates and 54% of MDR isolates) belonging to 15 different serotypes carried class 1 integrons that were transferred by conjugation in 17 isolates. Eleven distinct cassette arrangements were identified, with dfrA1-aadA1, dfrA17-aadA5, and dfrA12-orfF-aadA2 being the most prevalent and widely distributed ones. Atypical sul3-associated integrons were detected in 5 isolates of serotypes Rissen and Enteritidis. Moreover, the presence of integrons in the serotypes Kapemba, Mikawasima, and [9,12:Iv:i:-], of the estX-psp (linked to sul3) and aadA13-sat cassette arrangements in S. enterica, of extended-spectrum β-lactamases in S. Kapemba and S. Grumpensis, and of TQRD in S. Grumpensis is reported here for the first time.

Inhibition of beta-lactamase enzyme of Pseudomonas aeruginosa by clavulanic acid of Rumex vesicarius L.

This study plays an important role in solution of beta-lactam resistance problem. This aim has been achieved by clavulanic acid production as beta-lactamase inhibitor. Beta-lactam-resistant Pseudomonas aeruginosa possesses beta-lactams resistance machinery by secretion of extracellular beta-lactamase enzyme. The present study was begun with collection of eighty nosocomial bacterial isolates from Riyadh hospitals through one year. Nosocomial bacterial isolates were treated by amoxicillin (250 µg ml -1 ) to determine resistant isolates. Among them fifty amoxicillin-resistant isolates could be found with different minimum inhibitory concentrations (MICs). However, one isolate considers the most potent which has MIC over than 1000 µg ml -1 and also was identified by using polymerase chain reaction (PCR) technique as P. aeruginosa . Isolated gene of P. aeruginosa has 870 bp. The second phase of this study was begun with collection of six plants which are growing in Saudi Arabia, then were identified by professionals of Science College herbarium, King Saud University. These plants were extracted by methanol and then dried. These plants are Rhazya stricta , Calotropois procera , Maerua crassifolia , Haloxylon salicornicum , Rumex vesicarius L. and Lycium shawii . The plant extracts were tested to detect and determine their antagonistic effect for amoxicillin-resistant P. aeruginosa and of course for its beta-lactamase enzyme. Only one extract of R. vesicarius L. has antagonistic effect for beta-lactamase enzyme. Beta-lactamase inhibitor was purified and characterized as clavulanic acid by using nuclear magnetic resonance (NMR) spectroscopy analysis, infra-red (IR) spectroscopy analysis and elemental analysis. Eventually, amoxicillin-resistant P. aeruginosa was inhibited by amoxicillin-clavulanic acid combination at 125 µg ml -1 and 128 mg ml -1 , respectively.

Amoxicillin therapy of poultry flocks: effect upon the selection of amoxicillin-resistant commensal Campylobacter spp.

The aim of this study was to investigate the effect of amoxicillin therapy of poultry flocks upon the persistence of commensal Campylobacter spp. and the incidence of antibiotic resistance. Four poultry flocks naturally colonized with Campylobacter were treated with amoxicillin and monitored before, during and up to 4 weeks post-treatment. The numbers of Campylobacter were determined and the isolates speciated and typed by flaA short variable region (SVR) sequence analysis and PFGE. The susceptibility of the isolates to antibiotics, presence of the Cj0299 gene encoding a beta-lactamase and beta-lactamase production (nitrocefin hydrolysis) were also determined. Amoxicillin-resistant Campylobacter were isolated from Flock 1 before and during treatment, but Campylobacter were not detected afterwards. Flock 2 was colonized by amoxicillin-susceptible strains throughout sampling. No amoxicillin-resistant isolates arose during or after treatment. Flock 3 contained amoxicillin-susceptible and -resistant types pre-treatment. Resistant isolates were detected during treatment, while antibiotic-susceptible isolates re-emerged at 3 weeks post-treatment. All Campylobacter isolates from Flock 4 were amoxicillin resistant, irrespective of sampling time. All but one of the 82 amoxicillin-resistant (MICs 16 to >128 mg/L) Campylobacter jejuni and Campylobacter coli tested for the presence of Cj0299 carried the gene and all of these produced beta-lactamase. Co-amoxiclav remained active against amoxicillin-resistant isolates. Amoxicillin therapy had little effect on the numbers of amoxicillin-resistant commensal Campylobacter except for one flock where amoxicillin-resistant Campylobacter temporarily dominated. Amoxicillin therapy did not select amoxicillin-resistant isolates from a previous susceptible strain. Co-amoxiclav remained active against amoxicillin-resistant isolates.

Antibiotic Resistance in Haemophilus influenzae Decreased, except for β-Lactamase-Negative Amoxicillin-Resistant Isolates, in Parallel with Community Antibiotic Consumption in Spain from 1997 to 2007

The susceptibility to 14 antimicrobial agents and the mechanisms of aminopenicillin resistance were studied in 197 clinical isolates of Haemophilus influenzae--109 isolated in 2007 (study group) and 88 isolated in 1997 (control group). Community antibiotic consumption trends were also examined. H. influenzae strains were consecutively isolated from the same geographic area, mostly from respiratory specimens from children and adults. Overall, amoxicillin resistance decreased by 8.4% (from 38.6 to 30.2%). Beta-lactamase production decreased by 15.6% (from 33 to 17.4%, P = 0.01), but amoxicillin resistance without beta-lactamase production increased by 7.1% (from 5.7 to 12.8%). All beta-lactamase-positive isolates were TEM-1, but five different promoter regions were identified, with Pdel being the most prevalent in both years, and Prpt being associated with the highest amoxicillin resistance. A new promoter consisting of a double repeat of 54 bp was detected. Community consumption of most antibiotics decreased, as did the geometric means of their MICs, but amoxicillin-clavulanic acid and azithromycin consumption increased by ca. 60%. For amoxicillin-clavulanic acid, a 14.2% increase in the population with an MIC of 2 to 4 microg/ml (P = 0.02) was observed; for azithromycin, a 21.2% increase in the population with an MIC of 2 to 8 microg/ml (P = 0.0005) was observed. In both periods, the most common gBLNAR (i.e., H. influenzae isolates with mutations in the ftsI gene as previously defined) patterns were IIc and IIb. Community consumption of trimethoprim-sulfamethoxazole decreased by 54%, while resistance decreased from 50 to 34.9% (P = 0.04). Antibiotic resistance in H. influenzae decreased in Spain from 1997 to 2007, but surveillance should be maintained since new forms of resistances may be developing.

Antimicrobial susceptibility of Haemophilus influenzae strains and antibiotics usage patterns in pediatric outpatients: Results from a children's hospital in China (2000–2004)

To investigate the nasopharyngeal carriage and antimicrobial susceptibility of H. influenzae among children younger than 5 years old and to assess antibiotics usage patterns in the outpatient department of Beijing Children's Hospital from 2000 to 2004. From 2000 to 2004, At least 100 strains of H. influenzae were isolated from the pediatric patients who were younger than 5 years and who presented with symptoms of acute upper respiratory tract infections during February to May in each of the study years. Antimicrobial susceptibilities were determined; and antibiotics usage was expressed as defined daily dose (DDD)/100 patient days. The overall nasopharyngeal carriage rate of H. influenzae is 26.3% (562/2,137) in children younger than 5 years old with acute upper respiratory tract infection. The percentage of ampicillin-resistant isolates ranges from 4.0% (4/100) to 14.3% (17/119) from 2000 to 2004. All the ampicillin-resistant isolates are beta-lactamase producers. More than 80% of the isolates are susceptible to amoxicillin, cefaclor, and chloramphenicol; whereas, almost all (99-100%) of the isolates are sensitive to amoxicillin/clavulanic acid, ceftriaxone, and cefuroxime. For antibiotics utilization, macrolides are the predominantly used antibiotics, followed by cephalosporins and penicillins among pediatric patients in the outpatient department during the study period. All amoxicillin-resistant isolates of H. influenzae are producing beta-Lactamase; and the rates of amoxicillin-resistant isolates are increasing over time. Amoxicillin/clavulanic acid and cephalosporins are highly sensitive to H. influenzae isolated from Chinese pediatric patients. Macrolides are the most used antibiotics in the outpatient department during the study period.

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.

Helicobacter pylori amoxicillin heteroresistance due to point mutations in PBP-1A in isogenic isolates

To investigate the Helicobacter pylori amoxicillin resistance rate, the occurrence of heteroresistance, and their related molecular mechanisms. Eighty-seven H. pylori-positive patients were included: 45/87 with single biopsy and 42/87 with multiple biopsies. MICs were determined, and sequencing analysis of pbp1A gene and the variable regions of seven hop porins was performed in resistant and susceptible isolates. Clonal relationships were determined by lspA-glmM-RFLP and by random amplification of polymorphic DNA-PCR. An isogenic amoxicillin-susceptible isolate was transformed with pbp1A PCR products from the resistant isolates. Amoxicillin-resistant (MIC 2 mg/L) and amoxicillin-susceptible (MIC 0.06 mg/L) isolates, belonging to the same strain, were observed in different biopsies in one patient (inter-niche heteroresistance). Isolates from the remaining patients were amoxicillin-susceptible. Sequencing analysis of the pbp1A of two amoxicillin-resistant isolates and their susceptible partners revealed the same two point mutations: (i) in the third PBP motif of the resistant isolates (C1667G); (ii) a nonsense mutation at the 3' end of the gene. Replacement of pbp1A of a susceptible isolate by pbp1A from a resistant isolate increased the transformants MICs (2 mg/L). A similar MIC was observed when a pbp1A DNA fragment including both point mutations was transformed. Transfer of the smallest fragment (C1667G region only) yielded slightly lower MICs (0.5-1 mg/L). Identical hop gene sequences were observed in paired susceptible and resistant isolates. A low resistance rate was observed. However, inter-niche heteroresistance could hinder amoxicillin resistance detection when only one biopsy is obtained. Alteration in PBP-1A seems to be enough to reach an MIC of 2 mg/L in our resistant isolates.

Correlation between Substitutions in Penicillin‐Binding Protein 1 and Amoxicillin Resistance in Helicobacter pylori

The correlation between the substitutions of penicillin-binding protein 1 (PBP1) and amoxicillin resistance was studied for the determination of the substitutions in PBP1 which confer amoxicillin resistance in Helicobacter pylori. By the comparison of the amino acid sequences of PBP1 in the amoxicillinresistant (n=3), low-susceptible (n=3), and susceptible (n=13) H. pylori isolates, the substitution Asn562-->Tyr, which is adjacent to KTG motif (555-557), was common and specific to amoxicillin-resistant H. pylori. Additionally, all amoxicillin-resistant isolates had multiple substitutions such as Ser414-->Arg in the transpeptidase region of PBP1 of H. pylori. Furthermore all transformants obtained by the natural transformation using the pbp1 genes of amoxicillin-resistant H. pylori isolates had multiple substitutions including Asn562-->Tyr. These results suggest that multiple amino acid substitutions in the transpeptidase region of PBP1 are closely related to amoxicillin resistance in H. pylori.

The relative frequency of intraspecific lateral gene transfer of penicillin binding proteins 1a, 2b, and 2x, in amoxicillin resistant Streptococcus pneumoniae

Evidence exists for both interspecific and intraspecific recombination (lateral gene transfer; LGT) involving Streptococcus pneumoniae pbp (penicillin binding protein) loci. LGT of capsular genes, or serotype switching, is also know to occur between S. pneumoniae of different serotype. It is not clear whether intraspecific pbp LGT is relatively common, whether there is a difference in the relative frequency of intraspecific LGT of different pbps, and whether serotype switching is more or less frequent than pbp LGT. The purpose of this study was to use comparative evolutionary biology analysis of 216 international clinical S. pneumoniae isolates, from the Alexander Project collection, to gain insight on these issues, as well as the possible role they might be playing in spreading amoxicillin resistance. All 216 isolates were genotyped using MLST and complete or nearly complete sequences for pbp1a, pbp2b, and pbp2x were determined. Amoxicillin MICs were available for each isolate. pbps were genotyped using phylogenetics and two or more pbp types within a MLST sequence type (ST) or clonal complex were taken as putative cases of pbp LGT; these hypotheses were statistically evaluated using the approximately unbiased (AU) test. Serotypes were determined for 171 of these isolates and the minimum number of switching events necessary to explain the serotype phenotypes for each of the STs and clonal complexes were evaluated. The majority (78%) of the amoxicillin resistant isolates were comprised in 5 clonal complexes. The relative frequency of pbp LGT was greatest for pbp2b and 2x (minimum of 10.2 and 7.8%, respectively, of the isolates consistent with the LGT hypothesis), followed by 1a (3.9%). Serotype switching was more frequent than intraspecific pbp LGT (33% of isolates consistent with serotype switching hypothesis). Although intraspecific LGT of pbps is occurring and has played a role in the spread of amoxicillin resistance in S. pneumoniae, clonal dissemination appears to be more significant.

Longitudinal European surveillance study of antibiotic resistance of Haemophilus influenzae

We assessed the current resistance rates of Haemophilus influenzae against beta-lactams and other agents in Europe and compared the results with those of our previously performed surveillance study. MICs of the antibiotics were determined using broth microdilution. The penicillin-binding domain of PBP3 of beta-lactamase (BL)-negative, amoxicillin-resistant (BLNAR) isolates was sequenced. The percentage of BL-positive and BLNAR strains ranged from 0% to 17.6% and 0% to 33.9%, respectively. Compared with 1997/98 and 2002/03, the overall percentage of strains non-susceptible to amoxicillin decreased from 19.8% and 23.3%, respectively, to 16.4% in 2004/05. The percentage of BL-producing strains decreased from 11.0% and 13.7%, respectively, to 7.6%, whereas the number of BLNAR strains remained stable (8.8% and 9.6%, respectively, versus 8.8% in 2004/05). Comparison of penicillin binding protein (PBP) 3B gene sequences between BLNAR and susceptible strains revealed novel amino acid mutations. In spite of large inter-regional differences, the overall resistance of H. influenzae to amoxicillin in Europe seems to decline due to a decreasing number of BL-producing strains, whereas the overall percentage of BLNAR strains seems relatively constant.

北部九州・山口地区で分離された<i>Escherichia coli</i> O157およびVerotoxin産生<i>Escherichia coli</i>の性状および薬剤感受性とその耐性機序について

The purpose of this study was to investigate the biochemical characteristics and antimicrobial susceptibility of Escherichia (E.) coli O157 and verotoxin-producing E. coli isolates from the Northern Kyushu Island and Yamaguchi area of Japan. A total of 54 isolates- 50 E. coli O157, 3 verotoxin-producing E. coli O26 and 1 verotoxin-producing E. coli O111 - were used in this study. Regarding H antigen, H7 type in E. coli O157 accounted for 98% (49/50), and residual 1 strain of E. coli O157 was untypable H type. Two of 3 E. coli O26 isolates were H11 type, residual 1 strain of E. coli O26 was untypable H type, and E. coli O111 isolate was non-motile strain. All 54 isolates were susceptible to cephems, fosfomycin, kanamycin, amikacin and co-trimoxazole. Tetracycline-resistant isolates existed in 13 of all 54 isolates, 5 of those 13 isolates had tetA, and the other 7 isolates had tetB. Eight amoxicillin-resistant isolates had TEM-1 beta-lactamase. Four of the 5 isolates that had tetA also had TEM-1 beta-lactamase. Nalidixic acid and 6 fluoroquinolone used had no insensitive or resistant isolates. Kanamycin-resistant isolates, fosfomycin- and nalidixic acid-insensitive isolates have been reported, so we must notice the antibiogram of such strains. It is important that the surveillance of antimicrobial susceptibility of enterohemorragic E. coli should be continued after this.