High cephalosporin resistance due to amino acid substitutions in PBP1A and PBP2X in a clinical isolate of group B Streptococcus

Streptococcus agalactiae(group B streptococcus) isolates (n= 443) obtained from Japanese adults with invasive infections between April 2010 and March 2013 were analyzed for capsular serotype, multilocus sequence type (ST), antibiotic susceptibility, and resistance genes. Among these cases, bacteremia without primary focus was the most common variety of infection (49.9%), followed by cellulitis (12.9%) and pneumonia (9.0%). Concerning patient age (18 to 59, 60 to 69, 70 to 79, 80 to 89, and 90 years old or older), the incidence of pneumonia increased in patients in their 70s and 80s (P< 0.001), while younger patients (18 to 59 and 60 to 69 years old) were more likely to have abscesses (P< 0.05). The mortality rate was 10.2% for all ages. The most common capsular serotype was Ib (39.5%), followed by V (16.0%), III (13.8%), VI (9.5%), and Ia (8.6%). The main ST of serotype Ib strains was ST10, which belonged to clonal complex 10 (88.0%). The predominant clonal complexes of serotypes V and III, respectively, were 1 (78.9%) and 19 (75.4%). Among these isolates, 9 strains (2.0%) were identified as group B streptococci with reduced penicillin susceptibility, reflecting amino acid substitutions in penicillin-binding protein 2X (PBP2X). In addition, 19.2% of all strains possessedmef(A/E),erm(A), orerm(B) genes, which mediate macrolide resistance, while 40.2% of strains were resistant to quinolones resulting from amino acid substitutions in GyrA and ParC. Our data argue strongly for the continuous surveillance of microbial characteristics and judicious antibiotic use in clinical practice.

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AmpC β-Lactamase in an Escherichia coli Clinical Isolate Confers Resistance to Expanded-Spectrum Cephalosporins

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.10.4050-4053.2004 ◽

2004 ◽

Vol 48 (10) ◽

pp. 4050-4053 ◽

Cited By ~ 16

Author(s):

Hedi Mammeri ◽

Hasan Nazic ◽

Thierry Naas ◽

Laurent Poirel ◽

Sophie Léotard ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Clinical Isolate ◽

Biochemical Analysis ◽

Amino Acid Substitutions ◽

E Coli ◽

Extended Spectrum

ABSTRACT Cloning, sequencing, and biochemical analysis identified a novel AmpC-type β-lactamase conferring resistance to extended-spectrum cephalosporins in an Escherichia coli clinical isolate. This enzyme, exhibiting 14 amino acid substitutions compared to a reference AmpC cephalosporinase of E. coli, hydrolyzed ceftazidime and cefepime significantly.

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Classification of group B streptococci with reduced -lactam susceptibility (GBS-RBS) based on the amino acid substitutions in PBPs

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkv022 ◽

2015 ◽

Cited By ~ 3

Author(s):

K. Kimura ◽

N. Nagano ◽

Y. Arakawa

Keyword(s):

Amino Acid ◽

Amino Acid Substitutions ◽

Group B Streptococci ◽

Group B

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TEM-121, a Novel Complex Mutant of TEM-Type β-Lactamase from Enterobacter aerogenes

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.12.4528-4531.2004 ◽

2004 ◽

Vol 48 (12) ◽

pp. 4528-4531 ◽

Cited By ~ 22

Author(s):

Laurent Poirel ◽

Hedi Mammeri ◽

Patrice Nordmann

Keyword(s):

Amino Acid ◽

Kinetic Parameters ◽

Clavulanic Acid ◽

Clinical Isolate ◽

Amino Acid Change ◽

Enterobacter Aerogenes ◽

Kinetic Properties ◽

Amino Acid Substitutions ◽

Extended Spectrum ◽

Novel Complex

ABSTRACT Enterobacter aerogenes clinical isolate LOR was resistant to penicillins and ceftazidime but susceptible to cefuroxime, cephalothin, cefoxitin, cefotaxime, ceftriaxone, and cefepime. PCR and cloning experiments from this strain identified a novel TEM-type β-lactamase (TEM-121) differing by five amino acid substitutions from β-lactamase TEM-2 (Glu104Lys, Arg164Ser, Ala237Thr, Glu240Lys, and Arg244Ser) and by only one amino acid change from the extended-spectrum β-lactamase (ESBL) TEM-24 (Arg244Ser), with the last substitution also being identified in the inhibitor-resistant β-lactamase IRT-2. Kinetic parameters indicated that TEM-121 hydrolyzed ceftazidime and aztreonam (like TEM-24) and was inhibited weakly by clavulanic acid and strongly by tazobactam. Thus, TEM-121 is a novel complex mutant TEM β-lactamase (CMT-4) combining the kinetic properties of an ESBL and an inhibitor-resistant TEM enzyme.

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Precise genome editing using a CRISPR-Cas9 method highlights the role of CoERG11 amino acid substitutions in azole resistance in Candida orthopsilosis

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkz204 ◽

2019 ◽

Vol 74 (8) ◽

pp. 2230-2238 ◽

Cited By ~ 9

Author(s):

Florent Morio ◽

Lisa Lombardi ◽

Ulrike Binder ◽

Cédric Loge ◽

Estelle Robert ◽

...

Keyword(s):

Amino Acid ◽

Genome Editing ◽

Clinical Isolate ◽

Antifungal Susceptibility ◽

Azole Resistance ◽

Amino Acid Substitutions ◽

Fluconazole Resistance ◽

Candida Orthopsilosis

AbstractBackgroundAzoles are one of the main antifungal classes for the treatment of candidiasis. In the current context of emerging drug resistance, most studies have focused on Candida albicans, Candida glabrata or Candida auris but, so far, less is known about the underlying mechanisms of resistance in other species, including Candida orthopsilosis.ObjectivesWe investigated azole resistance in a C. orthopsilosis clinical isolate recovered from a patient with haematological malignancy receiving fluconazole prophylaxis.MethodsAntifungal susceptibility to fluconazole was determined in vitro (CLSI M27-A3) and in vivo (in a Galleria mellonella model of invasive candidiasis). The CoERG11 gene was then sequenced and amino acid substitutions identified were mapped on the predicted 3D structure of CoErg11p. A clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) genome-editing strategy was used to introduce relevant mutations into a fluconazole-susceptible C. orthopsilosis isolate.ResultsCompared with unrelated C. orthopsilosis isolates, the clinical isolate exhibited both in vitro and in vivo fluconazole resistance. Sequencing of the CoERG11 gene identified several amino acid substitutions, including two possibly involved in fluconazole resistance (L376I and G458S). Both mutations mapped close to the active site of CoErg11p. Engineering these mutations in a different genetic background using CRISPR-Cas9 demonstrated that G458S, but not L376I, confers resistance to fluconazole and voriconazole.ConclusionsOur data show that the G458S amino acid substitution in CoERG11p, but not L376I, contributes to azole resistance in C. orthopsilosis. In addition to highlighting the potential of CRISPR-Cas9 technology for precise genome editing in the field of antifungal resistance, we discuss some points that are critical to improving its efficiency.

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CTX-M-5, a Novel Cefotaxime-Hydrolyzing β-Lactamase from an Outbreak of Salmonella typhimuriumin Latvia

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.8.1980 ◽

1998 ◽

Vol 42 (8) ◽

pp. 1980-1984 ◽

Cited By ~ 93

Author(s):

Patricia A. Bradford ◽

Youjun Yang ◽

Daniel Sahm ◽

Ilze Grope ◽

Dace Gardovska ◽

...

Keyword(s):

Amino Acid ◽

Salmonella Typhimurium ◽

Nucleotide Sequencing ◽

Amino Acid Substitutions ◽

Gene Encoding ◽

Extended Spectrum ◽

Cephalosporin Resistance ◽

Resistant Strains ◽

Lactamase Gene ◽

Gene Expressing

ABSTRACT At a children’s hospital in Riga, Latvia, isolates identified asSalmonella typhimurium were found to be resistant to expanded-spectrum cephalosporins. Two of the resistant strains were analyzed for the mechanism of cephalosporin resistance. Isoelectric focusing revealed a common β-lactamase with a pI of 8.8. In addition, one of the strains produced a pI 7.6 β-lactamase. A transconjugant producing only the pI 7.6 enzyme was susceptible to expanded-spectrum cephalosporins; therefore, this enzyme was most likely SHV-1. Transformants producing only the pI 8.8 β-lactamase were resistant to cefotaxime and aztreonam but were susceptible or intermediate to ceftazidime. A substrate profile determined spectrophotometrically with purified enzyme revealed potent activity against cefotaxime, with a relative k cat value of 95 (benzylpenicillin equal to 100). The enzyme showed lower relativek cat values for ceftazidime (3.3) and aztreonam (9.3). In addition, the enzyme was inhibited by clavulanate, sulbactam and tazobactam, with 50% inhibitory concentrations of 19, 100, and 3.4 nM, respectively. These results indicated the presence of an unusual extended-spectrum β-lactamase. The gene expressing the pI 8.8 β-lactamase was cloned. Nucleotide sequencing revealed a β-lactamase gene that differs from the gene encoding CTX-M-2, which also originated from S. typhimurium, by 11 nucleotides, 4 of which result in amino acid substitutions: Ala27Thr, Val230Gly, Glu254Ala, and Ile278Val. These results indicated the presence of a novel extended-spectrum β-lactamase, designated CTX-M-5, that specifically confers resistance to cefotaxime.

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First Molecular Characterization of Group B Streptococci with Reduced Penicillin Susceptibility

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00185-08 ◽

2008 ◽

Vol 52 (8) ◽

pp. 2890-2897 ◽

Cited By ~ 154

Author(s):

Kouji Kimura ◽

Satowa Suzuki ◽

Jun-ichi Wachino ◽

Hiroshi Kurokawa ◽

Kunikazu Yamane ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Substitutions ◽

Group B Streptococci ◽

Allelic Exchange ◽

Specific Serum ◽

First Choice ◽

Invasive Diseases ◽

Group B ◽

First Time

ABSTRACT Group B streptococci (GBS; Streptococcus agalactiae) are the leading cause of neonatal invasive diseases and are also important pathogens for adults. Penicillins are the drugs of first choice for the treatment of GBS infections, since GBS have been regarded to be uniformly susceptible to penicillins so far. Here we characterize the first strains of GBS with reduced penicillin susceptibility (PRGBS) identified in Japan. Fourteen PRGBS strains were clinically isolated from the sputa of elderly patients from 1995 to 2005; and the MICs of penicillin, oxacillin, and ceftizoxime ranged from 0.25 to 1 μg/ml, 2 to 8 μg/ml, and 4 to 128 μg/ml, respectively. Moreover, some strains were also insusceptible to ampicillin, cefazolin, cefepime, and cefotaxime. All the PRGBS isolates tested possessed a few amino acid substitutions adjacent to the conserved SSN and KSG motifs (amino acids 402 to 404 and 552 to 554, respectively) of PBP 2X, and the amino acid substitutions could be classified into two types, Q557E and V405A. Western blotting analysis of the 14 clinical isolates with anti-PBP 2X-specific serum suggested that the amount of PBP 2X among the 14 PRGBS isolates was reduced, although the 2 ATCC strains produced a significant amount of PBP 2X. The introduction of PRGBS-derived PBP 2X genes into penicillin-susceptible strains through allelic exchange elevated their penicillin insusceptibility, suggesting that these altered PBP 2X genes are responsible for the penicillin insusceptibility in PRGBS strains. In this study, we characterized for the first time PRGBS strains on a molecular basis, although several reports have so far mentioned the existence of β-lactam-insusceptible GBS from a phenotypic standpoint.

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NDM-12, a Novel New Delhi Metallo-β-Lactamase Variant from a Carbapenem-Resistant Escherichia coli Clinical Isolate in Nepal

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03355-14 ◽

2014 ◽

Vol 58 (10) ◽

pp. 6302-6305 ◽

Cited By ~ 27

Author(s):

Tatsuya Tada ◽

Basudha Shrestha ◽

Tohru Miyoshi-Akiyama ◽

Kayo Shimada ◽

Hiroshi Ohara ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Clinical Isolate ◽

Urine Sample ◽

Enzymatic Activities ◽

Amino Acid Substitutions ◽

New Delhi ◽

Content Type ◽

Carbapenem Resistant

ABSTRACTA novel New Delhi metallo-β-lactamase variant, NDM-12, was identified in a carbapenem-resistantEscherichia coliclinical isolate obtained from a urine sample from a patient in Nepal. NDM-12 differed from NDM-1 by two amino acid substitutions (M154L and G222D). The enzymatic activities of NDM-12 against β-lactams were similar to those of NDM-1, although NDM-12 showed lowerkcat/Kmratios for all β-lactams tested except doripenem. TheblaNDM-12gene was located in a plasmid of 160 kb.

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Structural and Genetic Diversity of Group B Streptococcus Capsular Polysaccharides

Infection and Immunity ◽

10.1128/iai.73.5.3096-3103.2005 ◽

2005 ◽

Vol 73 (5) ◽

pp. 3096-3103 ◽

Cited By ~ 145

Author(s):

Michael J. Cieslewicz ◽

Donald Chaffin ◽

Gustavo Glusman ◽

Dennis Kasper ◽

Anup Madan ◽

...

Keyword(s):

Amino Acid ◽

Dna Sequences ◽

Antigenic Variation ◽

Group B Streptococcus ◽

Human Infection ◽

Amino Acid Sequences ◽

Capsular Polysaccharides ◽

En Bloc ◽

Synthetic Enzymes ◽

Group B

ABSTRACT Group B Streptococcus (GBS) is an important pathogen of neonates, pregnant women, and immunocompromised individuals. GBS isolates associated with human infection produce one of nine antigenically distinct capsular polysaccharides which are thought to play a key role in virulence. A comparison of GBS polysaccharide structures of all nine known GBS serotypes together with the predicted amino acid sequences of the proteins that direct their synthesis suggests that the evolution of serotype-specific capsular polysaccharides has proceeded through en bloc replacement of individual glycosyltransferase genes with DNA sequences that encode enzymes with new linkage specificities. We found striking heterogeneity in amino acid sequences of synthetic enzymes with very similar functions, an observation that supports horizontal gene transfer rather than stepwise mutagenesis as a mechanism for capsule variation. Eight of the nine serotypes appear to be closely related both structurally and genetically, whereas serotype VIII is more distantly related. This similarity in polysaccharide structure strongly suggests that the evolutionary pressure toward antigenic variation exerted by acquired immunity is counterbalanced by a survival advantage conferred by conserved structural motifs of the GBS polysaccharides.

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