Penicillin-resistant Clinical Isolates Research Articles

PBP2a in β-Lactam-Resistant Laboratory Mutants and Clinical Isolates: Disruption Versus Reduced Penicillin Affinity.

Alterations in PBP2a have been recognized in cefotaxime-resistant laboratory mutants and β-lactam-resistant clinical isolates of Streptococcus pneumoniae. DNA sequencing revealed fundamental differences between these two settings. Internal stop codons in pbp2a occurred in all three laboratory mutants analyzed, caused by a mutation in pbp2a of mutant C604, and tandem duplications within pbp2a resulting in premature stop codons in another two mutants C403 and C406. In contrast, mosaic PBP2a genes were observed in several penicillin-resistant clinical isolates from South Africa, the Czech Republic, Hungary, and in the clone Poland23F-16, with sequence blocks diverging from sensitive strains by over 4%. Most of these pbp2a variants except pbp2a from the South African strain contained sequences related to pbp2a of Streptococcus mitis B6, confirming that this species serves as reservoir for penicillin-resistance determinants.

Penicillin induces alterations in glutamine metabolism in Streptococcus pneumoniae

Penicillin is a bactericidal antibiotic that inhibits the synthesis of the peptidoglycan by targeting penicillin-binding proteins. This study aimed to assess through transcriptional profiling the stress response of S. pneumoniae strains after exposure to lethal penicillin concentrations to understand further the mode of action of penicillin. Two experimental designs (time-course and dose-response) were used for monitoring the effect of penicillin on the transcriptional profile. The expression of some genes previously shown to be modulated by penicillin was altered, including ciaRH, pstS and clpL. Genes of the glnRA and glnPQ operons were among the most downregulated genes in the three strains. These genes are involved in glutamine synthesis and uptake and LC-MS work confirmed that penicillin treatment increases the intracellular glutamine concentrations. Glutamine conferred a protective role against penicillin when added to the culture medium. Glutamine synthetase encoded by glnA catalyses the transformation of glutamate and ammonium into glutamine and its chemical inhibition by the inhibitor L-methionine sulfoximine is shown to sensitize S. pneumoniae to penicillin, including penicillin-resistant clinical isolates. In summary, a combination of RNA-seq and metabolomics revealed that penicillin interferes with glutamine metabolism suggesting strategies that could eventually be exploited for combination therapy or for reversal of resistance.

Genomic Analyses of DNA Transformation and Penicillin Resistance in Streptococcus pneumoniae Clinical Isolates

Alterations in penicillin-binding proteins, the target enzymes for β-lactam antibiotics, are recognized as primary penicillin resistance mechanisms in Streptococcus pneumoniae. Few studies have analyzed penicillin resistance at the genome scale, however, and we report the sequencing of S. pneumoniae R6 transformants generated while reconstructing the penicillin resistance phenotypes from three penicillin-resistant clinical isolates by serial genome transformation. The genome sequences of the three last-level transformants T2-18209, T5-1983, and T3-55938 revealed that 16.2 kb, 82.7 kb, and 137.2 kb of their genomes had been replaced with 5, 20, and 37 recombinant sequence segments derived from their respective parental clinical isolates, documenting the extent of DNA transformation between strains. A role in penicillin resistance was confirmed for some of the mutations identified in the transformants. Several multiple recombination events were also found to have happened at single loci coding for penicillin-binding proteins (PBPs) that increase resistance. Sequencing of the transformants with MICs for penicillin similar to those of the parent clinical strains confirmed the importance of mosaic PBP2x, -2b, and -1a as a driving force in penicillin resistance. A role in resistance for mosaic PBP2a was also observed for two of the resistant clinical isolates.

The 2011 Garrod Lecture: From penicillin-binding proteins to molecular epidemiology

In this review, based on my Garrod Lecture to the British Society for Antimicrobial Chemotherapy, I have given a brief outline of my career over the past 40 years, starting with research in the 1970s into the properties and functions of penicillin-binding proteins (PBPs), leading to the identification of the high molecular mass PBPs as the physiological targets of penicillin, and subsequent studies showing the emergence of low-affinity PBPs in penicillin-resistant clinical isolates by inter-species recombination and the generation of mosaic PBP genes. The studies of clinical isolates of gonococci, meningococci and pneumococci with PBP-mediated resistance to penicillin led to new interests in molecular epidemiology and the population and evolutionary biology of bacterial pathogens. The development (with colleagues) of multilocus sequence typing provided a method for the unambiguous characterization of bacterial strains that has proved to be very widely used, but the recent remarkable (and ongoing) developments in DNA sequencing technologies have provided the prospect of being able routinely to use whole genome sequences to characterize pathogen isolates. These developments will soon have major implications for diagnostic microbiology, outbreak investigations and our ability to follow the spread of strains of community-acquired and nosocomial pathogens at local, national and international levels. However, there are major barriers to be overcome, particularly with respect to how the avalanche of genome sequence data will be stored so that its transformative potential for molecular epidemiology and international public health are fully realized.

An Important Site in PBP2x of Penicillin-Resistant Clinical Isolates of Streptococcus pneumoniae : Mutational Analysis of Thr338

Penicillin-binding protein 2x (PBP2x) of Streptococcus pneumoniae represents a primary resistance determinant for beta-lactams, and low-affinity PBP2x variants can easily be selected with cefotaxime. Penicillin-resistant clinical isolates of S. pneumoniae frequently contain in their mosaic PBP2x the mutation T338A adjacent to the active site S337, and T338P as well as T338G substitutions are also known. Site-directed mutagenesis has now documented that a single point mutation at position T338 confers selectable levels of beta-lactam resistance preferentially to oxacillin. Despite the moderate impact on beta-lactam susceptibility, the function of the PBP2x mutants appears to be impaired, as can be documented in the absence of a functional CiaRH regulatory system, resulting in growth defects and morphological changes. The combination of low-affinity PBP2x and PBP1a encoded by mosaic genes is known to result in high cefotaxime resistance. In contrast, introduction of a mosaic pbp1a into the PBP2x(T338G) mutant did not lead to increased resistance. However, the mosaic PBP1a gene apparently complemented the PBP2x(T338G) defect, since Cia mutant derivatives grew normally. The data support the view that PBP2x and PBP1a interact with each other on some level and that alterations of both PBPs in resistant clinical isolates have evolved to ensure cooperation between both proteins.

The CiaRH System ofStreptococcus pneumoniaePrevents Lysis during Stress Induced by Treatment with Cell Wall Inhibitors and by Mutations inpbp2xInvolved in β-Lactam Resistance

The two-component signal-transducing system CiaRH of Streptococcus pneumoniae plays an important role during the development of beta-lactam resistance in laboratory mutants. We show here that a functional CiaRH system is required for survival under many different lysis-inducing conditions. Mutants with an activated CiaRH system were highly resistant to lysis induced by a wide variety of early and late cell wall inhibitors, such as cycloserine, bacitracin, and vancomycin, and were also less susceptible to these drugs. In contrast, loss-of-function CiaRH mutants were hypersusceptible to these drugs and were apparently unable to maintain a stationary growth phase in normal growth medium and under choline deprivation as well. Moreover, disruption of CiaR in penicillin-resistant mutants with an altered pbp2x gene encoding low-affinity PBP2x resulted in severe growth defects and rapid lysis. This phenotype was observed with pbp2x genes containing point mutations selected in the laboratory and with highly altered mosaic pbp2x genes from penicillin-resistant clinical isolates as well. This documents for the first time that PBP2x mutations required for development of beta-lactam resistance are functionally not neutral and are tolerated only in the presence of the CiaRH system. This might explain why cia mutations have not been observed in penicillin-resistant clinical isolates. The results document that the CiaRH system is required for maintenance of the stationary growth phase and for prevention of autolysis triggered under many different conditions, suggesting a major role for this system in ensuring cell wall integrity.

Synthesis and Antimicrobial Properties of 2‐(Benzylidene‐amino)‐benzo[d]isothiazol‐3‐ones.

AbstractFor Abstract see ChemInform Abstract in Full Text.

In vitro combined effect of co-amoxiclav concentrations achievable in serum after a 2000/125 mg oral dose, and polymorphonuclear neutrophils against strains of Streptococcus pneumoniae exhibiting decreased susceptibility to amoxicillin

The in vitro effect that the presence of components of non-specific immunity (serum plus polymorphonuclear neutrophils) has on the bactericidal activity of co-amoxiclav was explored against Streptococcus pneumoniae strains exhibiting an amoxicillin MIC ≥4 mg/L. Eight penicillin-resistant clinical isolates non-susceptible to co-amoxiclav with MICs of 4 (two strains), 8 (four strains) and 16 mg/L (two strains) were used. Values of MBC were identical to MIC values in all cases. Time-kill curves were performed with co-amoxiclav concentrations achievable in serum after a single oral dose administration of the new 2000/125 mg sustained-release formulation. Results were expressed as percentage of reduction of initial inocula after 3 h incubation. Control curves showed growth with no reduction of initial inocula. Against strains with MIC of 4 and 8 mg/L, the results obtained with the antibiotic alone or with the presence of factors of non-specific immunity were similar, with a weak combined effect due to the intrinsic activity of co-amoxiclav (reductions of initial inocula ranging from 70 to 99.16%). Against strains with MIC of 16 mg/L, the addition of PMN in the presence of serum increased the reduction of bacterial load provided by the aminopenicillin, even at sub-inhibitory concentrations (25.8% versus 51.1% at 0.5×MIC concentration − 8/0.5 mg/L). This combined activity against strains with an amoxicillin MIC of 16 mg/L which decreased the bacterial load may be important in preventing bacterial proliferation within the host and the transmission of resistant clones to others.

Synthesis and antimicrobial properties of 2-(benzylidene-amino)-benzo[ d]isothiazol-3-ones

The in vitro antimicrobial activity of 2-amino-benzo[ d]isothiazol-3-one and of several 2-arylideneamino derivatives carrying in the second position a substituted or unsubstituted aromatic ring or an arylalkenylidene moiety was determined by the broth dilution method against several strains selected to define their spectrum and potency. All the compounds demonstrated good antibacterial properties against Bacillus subtilis, streptococci, enterococci and staphylococci including penicillin-resistant clinical isolates. Several compounds showed excellent inhibitory properties against Streptococcus pyogenes, which is the most sensitive microorganism tested. Many benzisothiazolones exhibited good activity against Gram-negative Haemophilus influenzae. As regards antifungal activity, several of the tested compounds inhibited Saccharomyces cerevisiae at concentrations of 3–6 μg ml –1. In all cases the parent 2-amino-benzo[ d]isothiazol-3-one was the most effective agent, with minimum inhibitory concentration (MIC) values ranging from 0.07 to 6 μg ml –1. The results obtained indicate that most of these compounds are wide-spectrum antimicrobial substances and promising agents against penicillin-resistant staphylococci.

Alterations to penicillin-binding proteins 1A, 2B and 2X amongst penicillin-resistant clinical isolates of Streptococcus pneumoniae serotype 23F from the nasopharyngeal flora of children.

Various amino acid substitutions were identified in the three major penicillin-binding proteins (PBP1A, PBP2B and PBP2X) of eight clinical isolates of Streptococcus pneumoniae serotype 23F collected from children. The particular changes related to the level of penicillin resistance. Alterations were detected in an isolate with a penicillin MIC as low as 0.06 mg/L. These results confirm that the level of penicillin resistance in pneumococci reflects with sequential alterations of PBPs in clinical isolates.

Penicillin-binding proteins as resistance determinants in clinical isolates of Streptococcus pneumoniae.

Altered penicillin-binding proteins (PBPs) with reduced affinity for penicillin are encoded by mosaic genes in penicillin-resistant clinical isolates of Streptococcus pneumoniae. Generally, members of one bacterial clone contain the same mosaic gene. We report here on a serotype 19A clone of penicillin- and multiple-resistant S. pneumoniae prevalent in Hungary, members of which are exceptionally diverse in terms of PBP properties. The pbp2x gene of four 19A isolates was sequenced, and a distinct mosaic structure detected in each case. The pbp2x genes also differed from a homologous gene of a high-level penicillin-resistant S. mitis from Hungary. The contribution of PBPs to resistance development was studied on transformation experiments using the laboratory strain R6 as recipient, and PBP genes from the type 19A isolate Hu11. pbp2x and pbp2b function as primary resistance determinants for different beta-lactams. Secondary transformation with pbp1a increased the resistance level considerably for penicillins and cefotaxime. Chromosomal DNA of a high-level penicillin- and cefotaxime-resistant S. mitis from Hungary also transformed the R6 strain to increased resistance levels, and PBP2x and PBP2b functioned as primary resistance determinants as above. In contrast, high-level cefotaxime resistance appeared to be due to a low affinity PBP2a, indicating that this PBP can also function as a resistance determinant.

Mosaic pbpX genes of major clones of penicillin-resistant Streptococcus pneumoniae have evolved from pbpX genes of a penicillin-sensitive Streptococcus oralis.

Penicillin-resistant clinical isolates of Streptococcus pneumoniae contain mosaic penicillin-binding protein (PBP) genes that encode PBPs with decreased affinity for beta-lactam antibiotics. The mosaic blocks are believed to be the result of gene transfer of homologous PBP genes from related penicillin-resistant species. We have now identified a gene homologous to the pneumococcal PBP2x gene (pbpX) in a penicillin-sensitive Streptococcus oralis isolate M3 from South Africa that diverged by almost 20% from pbpX of penicillin-sensitive pneumococci, and a central sequence block of a mosaic pbpX gene of Streptococcus mitis strain NCTC 10712. In contrast, it differed by only 2-4% of the 1 to 1.5 kb mosaic block in pbpX genes of three genetically unrelated penicillin-resistant S. pneumoniae isolates, two of them representing clones of serotype 6B and 23F, which are prevalent in Spain and are also already found in other countries. With low concentrations of cefotaxime, transformants of the sensitive S. pneumoniae R6 strain could be selected containing pbpX genes from either S. mitis NCTC 10712 or S. oralis M3, demonstrating that genetic exchange can already occur between beta-lactam-sensitive species. These data are in agreement with the assumption that PBPs as penicillin-resistance determinants have evolved by the accumulation of point mutations in genes of sensitive commensal species.

Increase in Moderate Penicillin Resistance and Serogroup C in Meningococcal Strains Isolated in Spain. Is There Any Relationship?

Serogroup B Neisseria meningitidis is the main cause of meningococcal disease in Spain, but in recent years we have detected an increase in the prevalence of infection due to serogroup C meningococci. At the same time, the frequency of moderately penicillin-resistant (PenR) clinical isolates, which include greater numbers of serogroup C meningococci than do penicillin-susceptible (PenS) strains, has also been increasing. When we analyzed the prevalence of serogroups B and C in PenR and PenS meningococcal strains, we found a simultaneous increase in serogroup C strains and a decrease in serogroup B meningococci affecting both PenR and PenS isolates. To analyze this epidemiological change in Spain, we have applied serotyping, subtyping, and multilocus enzyme electrophoresis to serogroup C (PenR and PenS) strains. The two major serotypes were 2b and 2a in both groups (PenR and PenS), but our results suggested an association between serotype 2b and PenR strains. However, multilocus enzyme electrophoresis showed that 75% of the major serotypes belonged to the same electrophoretic type. It does not appear that a new clone distinct from those already established is contributing to the increase in serogroup C meningococci in Spain.

Regulation of the permeability of the gonococcal cell envelope by the mtr system

The mtrR gene of Neisseria gonorrhoeae controls the level of susceptibility to hydrophobic antibiotics and detergents. The mtrR gene was cloned and shown to encode a putative transcriptional repressor. The mtr region was homologous to the envCD and acrAB regions of Escherichia coli, which are also involved in susceptibility to hydrophobic compounds. A homologous repressor protein was encoded by a previously unrecognized open reading frame within both the envCD and acrAB regions. Deletion of mtrR resulted in increased resistance to antibiotics and detergents: the mtrR mutations in two penicillin-resistant clinical isolates resulted in a change of His-105 to Tyr. We propose that the mtrR repressor allows gonococci to regulate the permeability of its cell envelope in response to environmental signals, so that they can grow in the presence of toxic faecal lipids in the rectum as well as in the genital tract.

Mechanisim of resistance in penicillin-resistant clinical isolates of Streptococcus pneumoniae: Analysis of penicillin-binding proteins and the PBP 2 B gene

Mechanisim of resistance in penicillin-resistant clinical isolates of <I>Streptococcus pneumoniae</I>: Analysis of penicillin-binding proteins and the PBP 2 B gene

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.

Penicillin-binding proteins inStreptococcus mitis

The penicillin-binding protein (PBP) profiles of penicillin-susceptible and-resistant clinical isolates ofStreptococcus mitis varied even with strains with similar minimal inhibitory concentrations (MICs).S. mitis NCTC 10712 was used as a DNA recipient to investigate PBP alterations which could occur as a result of spontaneous mutation and intra- and interspecific transfer of penicillin resistance genes.S. mitis NCTC 10712 possesses seven major PBPs ranging in molecular mass from 49–82 kDa. TwoS. mitis and twoStreptococcus pneumoniae penicillin-resistant clinical isolates were used as donors in transformation experiments withS. mitis NCTC 10712 (MIC 0.03 μg/ml) as the recipient. Transformants with MICs greater than 1 μg/ml were obtained with bothS. mitis andS. pneumoniae donor DNA. Depending on the source of the donor DNA and level of resistance achieved, transformants showed reduced penicillin-binding affinities of PBPs 2, 3, 4, 5, and 6. The most consistent PBP alteration associated with increasing resistance inS. mitis NCTC 10712 was seen with PBP 3 (74 kDa).

Epidemiology and molecular basis of penicillin-resistant Neisseria meningitidis in Spain: a 5-year history (1985-1989).

Penicillin-resistant (penr) clinical isolates of Neisseria meningitidis, which do not produce beta-lactamase, were first identified in Spain in 1985; the frequency of their recovery, which has been increasing in the past few years, reached 20% in 1989. Serogrouping, determination of serotypes and subtypes, and multilocus enzyme electrophoresis of the penr strains showed an extensive diversity. Resistance is due, at least in part, to a decreased affinity of penicillin-binding protein (PBP) 2 for penicillin. Similar low-affinity forms of PBP 2 are also found in penr isolates of Neisseria lactamica, Neisseria polysaccharea, and Neisseria gonorrhoeae. Genetic transformation of an N. meningitidis type strain to low-level penicillin resistance with DNA from resistant meningococci and other Neisseria species resulted in transformants that possessed low-affinity forms of PBP 2. These altered forms of PBP 2 have been shown to arise from recombinational events that replace parts of the PBP 2 gene with the corresponding regions from the PBP 2 genes of commensal Neisseria species.

Interspecies recombinational events during the evolution of altered PBP 2x genes in penicillin‐resistant clinical isolates of Streptococcus pneumoniae

Penicillin resistance in pneumococci is due to the appearance of high molecular-weight penicillin-binding proteins (PBPs) that have reduced affinity for the antibiotic. We have compared the PBX 2x genes (pbpX) of one penicillin-susceptible and five penicillin-resistant clinical isolates of Streptococcus pneumoniae isolated from various parts of the world. All of the resistant isolates contained a low-affinity form of PBP 2x. The 2 kb region of the two penicillin-susceptible isolates differed at only eight nucleotide sites (0.4%) and resulted in one single amino acid difference in PBP 2x. In contrast, the sequences of the PBP 2x genes from the resistant isolates differed overall from those of the susceptible isolates at between 7 and 18% of nucleotide sites and resulted in between 27 and 86 amino acid substitutions in PBP 2x. The altered PBP 2x genes consisted of regions that were similar to those of susceptible strains (less than 3% diverged), alternating with regions that were very different (18-23% diverged). The presence of highly diverged regions within the PBP 2x genes of the resistant isolates contrasts with the uniformity of the sequences of the amylomaltase genes from the same isolates, and with the uniformity of the PBP 2x genes in the two susceptible isolates. It suggests that the altered PBP 2x genes have arisen by localized interspecies recombinational events involving the PBP 2x genes of closely related streptococci, as has been suggested to occur for altered PBP 2b genes (Dowson et al., 1989b). The PBP 2x genes from the resistant isolates could transform the susceptible strain R6 to increased levels of resistance to beta-lactam antibiotics, indicating that the altered forms of PBP 2x in the resistant isolates contribute to their resistance to penicillin.

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.