Evaluation of [89Zr]Zr-DFO-2Rs15d Nanobody for Imaging of HER2-Positive Breast Cancer.

One of the most aggressive forms of breast cancer involves the overexpression of human epidermal growth factor receptor 2 (HER2). HER2 is overexpressed in ∼25% of all breast cancers and is associated with increased proliferation, increased rates of metastasis, and poor prognosis. Treatment for HER2-positive breast cancer has vastly improved since the development of the monoclonal antibody trastuzumab (Herceptin) as well as other biological constructs. However, patients still commonly develop resistance, illustrating the need for newer therapies. Nanobodies have become an important focus for potential development as HER2-targeting imaging agents and therapeutics. Nanobodies have many favorable characteristics, including high stability in heat and nonphysiological pH, while maintaining their low-nanomolar affinity for their designed targets. Specifically, the 2Rs15d nanobody has been developed for targeting HER2 and has been evaluated as a diagnostic imaging agent for single-photon emission computed tomography (SPECT) and positron emission tomography (PET). While a construct of 2Rs15d with the positron emitter 68Ga is currently in phase I clinical trials, the only PET images acquired in preclinical or clinical research have been within 3 h postinjection. We evaluated our in-house produced 2Rs15d nanobody, conjugated with the chelator deferoxamine (DFO), and radiolabeled with 89Zr for PET imaging up to 72 h postinjection. [89Zr]Zr-DFO-2Rs15d demonstrated high stability in both phosphate-buffered saline (PBS) and human serum. Cell binding studies showed high binding and specificity for HER2, as well as prominent internalization. Our in vivo PET imaging confirmed high-quality visualization of HER2-positive tumors up to 72 h postinjection, whereas HER2-negative tumors were not visualized. Subsequent biodistribution studies quantitatively supported the significant HER2-positive tumor uptake compared to the negative control. Our studies fill an important gap in understanding the imaging and binding properties of the 2Rs15d nanobody at extended time points. As many therapeutic radioisotopes have single or multiday half-lives, this information will directly benefit the potential of the radiotherapy development of 2Rs15d for HER2-positive breast cancer patients.

Exposure of progressive immune dysfunction by SARS-CoV-2 mRNA vaccination in patients with chronic lymphocytic leukemia: A prospective cohort study.

BACKGROUND: Patients with chronic lymphocytic leukemia (CLL) have reduced seroconversion rates and lower binding antibody (Ab) and neutralizing antibody (NAb) titers than healthy individuals following Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) mRNA vaccination. Here, we dissected vaccine-mediated humoral and cellular responses to understand the mechanisms underlying CLL-induced immune dysfunction. METHODS AND FINDINGS: We performed a prospective observational study in SARS-CoV-2 infection-naïve CLL patients (n = 95) and healthy controls (n = 30) who were vaccinated between December 2020 and June 2021. Sixty-one CLL patients and 27 healthy controls received 2 doses of the Pfizer-BioNTech BNT162b2 vaccine, while 34 CLL patients and 3 healthy controls received 2 doses of the Moderna mRNA-1273 vaccine. The median time to analysis was 38 days (IQR, 27 to 83) for CLL patients and 36 days (IQR, 28 to 57) for healthy controls. Testing plasma samples for SARS-CoV-2 anti-spike and receptor-binding domain Abs by enzyme-linked immunosorbent assay (ELISA), we found that all healthy controls seroconverted to both antigens, while CLL patients had lower response rates (68% and 54%) as well as lower median titers (23-fold and 30-fold; both p < 0.001). Similarly, NAb responses against the then prevalent D614G and Delta SARS-CoV-2 variants were detected in 97% and 93% of controls, respectively, but in only 42% and 38% of CLL patients, who also exhibited >23-fold and >17-fold lower median NAb titers (both p < 0.001). Interestingly, 26% of CLL patients failed to develop NAbs but had high-titer binding Abs that preferentially reacted with the S2 subunit of the SARS-CoV-2 spike. Since these patients were also seropositive for endemic human coronaviruses (HCoVs), these responses likely reflect cross-reactive HCoV Abs rather than vaccine-induced de novo responses. CLL disease status, advanced Rai stage (III-IV), elevated serum beta-2 microglobulin levels (ß2m >2.4 mg/L), prior therapy, anti-CD20 immunotherapy (<12 months), and intravenous immunoglobulin (IVIg) prophylaxis were all predictive of an inability to mount SARS-CoV-2 NAbs (all p ≤ 0.03). T cell response rates determined for a subset of participants were 2.8-fold lower for CLL patients compared to healthy controls (0.05, 95% CI 0.01 to 0.27, p < 0.001), with reduced intracellular IFNγ staining (p = 0.03) and effector polyfunctionality (p < 0.001) observed in CD4+ but not in CD8+ T cells. Surprisingly, in treatment-naïve CLL patients, BNT162b2 vaccination was identified as an independent negative risk factor for NAb generation (5.8, 95% CI 1.6 to 27, p = 0.006). CLL patients who received mRNA-1273 had 12-fold higher (p < 0.001) NAb titers and 1.7-fold higher (6.5, 95% CI 1.3 to 32, p = 0.02) response rates than BNT162b2 vaccinees despite similar disease characteristics. The absence of detectable NAbs in CLL patients was associated with reduced naïve CD4+ T cells (p = 0.03) and increased CD8+ effector memory T cells (p = 0.006). Limitations of the study were that not all participants were subjected to the same immune analyses and that pre-vaccination samples were not available. CONCLUSIONS: CLL pathogenesis is characterized by a progressive loss of adaptive immune functions, including in most treatment-naïve patients, with preexisting memory being preserved longer than the capacity to mount responses to new antigens. In addition, higher NAb titers and response rates identify mRNA-1273 as a superior vaccine for CLL patients.

Inter- and intraspecific variation in mycotoxin tolerance: A study of four Drosophila species.

Many mycophagous Drosophila species have adapted to tolerate high concentrations of mycotoxins, an ability not reported in any other eukaryotes. Although an association between mycophagy and mycotoxin tolerance has been established in many Drosophila species, the genetic mechanisms of the tolerance are unknown. This study presents the inter- and intraspecific variation in the mycotoxin tolerance trait. We studied the mycotoxin tolerance in four Drosophila species from four separate clades within the immigrans-tripunctata radiation from two distinct locations. The effect of mycotoxin treatment on 20 isofemale lines per species was studied using seven gross phenotypes: survival to pupation, survival to eclosion, development time to pupation and eclosion, thorax length, fecundity, and longevity. We observed interspecific variation among four species, with D. falleni being the most tolerant, followed by D. recens, D. neotestacea, and D. tripunctata, in that order. The results also revealed geographical variation and intraspecific genetic variation in mycotoxin tolerance. This report provides the foundation for further delineating the genetic mechanisms of the mycotoxin tolerance trait.

Comprehensive mapping of SARS-CoV-2 peptide epitopes for development of a highly sensitive serological test for total and neutralizing antibodies.

Quantification of the anti-SARS-CoV-2 antibody response has proven to be a prominent diagnostic tool during the COVID-19 pandemic. Antibody measurements have aided in the determination of humoral protection following infection or vaccination and will likely be essential for predicting the prevalence of population level immunity over the next several years. Despite widespread use, current tests remain limited in part, because antibody capture is accomplished through the use of complete spike and nucleocapsid proteins that contain significant regions of overlap with common circulating coronaviruses. To address this limitation, a unique epitope display platform utilizing monovalent display and protease-driven capture of peptide epitopes was used to select high affinity peptides. A single round of selection using this strategy with COVID-19 positive patient plasma samples revealed surprising differences and specific patterns in the antigenicity of SARS-CoV-2 proteins, especially the spike protein. Putative epitopes were assayed for specificity with convalescent and control samples, and the individual binding kinetics of peptides were also determined. A subset of prioritized peptides was used to develop an antibody diagnostic assay that showed low cross reactivity while detecting 37% more positive antibody cases than a gold standard FDA EUA test. Finally, a subset of peptides were compared with serum neutralization activity to establish a 2 peptide assay that strongly correlates with neutralization. Together, these data demonstrate a novel phage display method that is capable of comprehensively and rapidly mapping patient viral antibody responses and selecting high affinity public epitopes for the diagnosis of humoral immunity.

SARS-CoV-2 mRNA vaccination exposes progressive adaptive immune dysfunction in patients with chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) patients have lower seroconversion rates and antibody titers following SARS-CoV-2 vaccination, but the reasons for this diminished response are poorly understood. Here, we studied humoral and cellular responses in 95 CLL patients and 30 healthy controls after two BNT162b2 or mRNA-2173 mRNA immunizations. We found that 42% of CLL vaccinees developed SARS-CoV-2-specific binding and neutralizing antibodies (NAbs), while 32% had no response. Interestingly, 26% were seropositive, but had no detectable NAbs, suggesting the maintenance of pre-existing endemic human coronavirus-specific antibodies that cross-react with the S2 domain of the SARS-CoV-2 spike. These individuals had more advanced disease. In treatment-naïve CLL patients, mRNA-2173 induced 12-fold higher NAb titers and 1.7-fold higher response rates than BNT162b2. These data reveal a graded loss of immune function, with pre-existing memory being preserved longer than the capacity to respond to new antigens, and identify mRNA-2173 as a superior vaccine for CLL patients.

Adhesion of Streptococcus pneumoniae to human airway epithelial cells exposed to urban particulate matter.

BACKGROUND: Epidemiologic studies report an association between pneumonia and urban particulate matter (PM) less than 10 microns (µm) in aerodynamic diameter (PM(10)). Streptococcus pneumoniae is a common cause of bacterial pneumonia worldwide. To date, the mechanism whereby urban PM enhances vulnerability to S pneumoniae infection is unclear. Adhesion of S pneumoniae to host cells is a prerequisite for infection. Host-expressed proteins, including the receptor for platelet-activating factor (PAFR), are co-opted by S pneumoniae to adhere to lower airway epithelial cells. OBJECTIVES: To define whether inhalable urban PM enhances the adhesion of S pneumoniae to airway epithelial cells. METHODS: A549 cells were cultured with PM(10) from Leicester (United Kingdom [UK]) and PM(10) and PM less than 2.5 µm in aerodynamic diameter (PM(2.5)) from Accra (Ghana), then infected with S pneumoniae strain D39. Pneumococcal adhesion to human primary bronchial epithelial cells was also assessed. Bacterial adhesion was determined by quantitative culture and confocal microscopy. The role of oxidative stress was assessed by N-acetyl cysteine, and the role of PAFR was assessed by mRNA transcript level, receptor expression, and receptor blocking. RESULTS: PM(10) (UK) increased S pneumoniae adhesion to both A549 airway epithelial cells and human primary bronchial epithelial cells. PM(10) (Ghana) and PM(2.5) (Ghana) also increased adhesion. Culture of A549 cells by PM(10) (UK) increased PAFR mRNA transcript level and PAFR expression. PM(10) (UK)-stimulated adhesion to A549 cells was attenuated by a PAFR blocker and N-acetyl cysteine. CONCLUSION: Urban PM increases adhesion of S pneumoniae to human airway epithelial cells. PM-stimulated adhesion is mediated by oxidative stress and PAFR.

Streptococcus pneumoniae isolates expressing a capsule with epitopes of both serotypes 6A and 6B.

Four Streptococcus pneumoniae isolates expressing both 6A and 6B capsular serotypes were detected by a multiplex immunoassay. The sequence of WciP, a GT2-family glycosyltransferase, indicates that point mutation has compromised linkage specificity, allowing two alternative oligosaccharides to be synthesized. This finding highlights that mutation as well as recombination can mediate serotype change.

Mutation frequency in antibiotic-resistant and -susceptible isolates of Streptococcus pneumoniae.

Development of multiple antibiotic resistance in Streptococcus pneumoniae typically involves either mutation or transformation at several well-separated chromosomal loci. We postulated that this series of genetic events would be more likely to occur in organisms with deficient DNA repair mechanisms. Investigation of 27 antibiotic-resistant or -susceptible clinical isolates of S. pneumoniae revealed a broad range of mutation frequencies, but no isolate was as mutable as a mismatch repair (MMR)-deficient laboratory isolate. No correlation was observed between antibiotic resistance and higher mutation frequency. Examination of a further 180 clinical isolates using a newly developed rapid screen method also failed to identify any isolates with a mutation frequency as high as the MMR-deficient control strain. We argue that there is currently no clear evidence of a distinct population of mutators among clinical pneumococci.

Plasmids imparting sulfonamide resistance in Escherichia coli: implications for persistence.

Sulfonamide resistance remains prevalent among clinical isolates of Escherichia coli in the United Kingdom, despite a dramatic (>97%) national decline in the rate of prescription of sulfonamides in the 1990s. To investigate potential mechanisms accounting for this persistence, we characterized plasmids carrying sul2, the most prevalent determinant of sulfonamide resistance. Among 33 conjugative and 5 nonconjugative plasmids carrying sul2, resistance to other antimicrobial agents was common, but the spectrum of resistance profiles was diverse: 82%, 74%, and 45% carried resistance to ampicillin, streptomycin, and trimethoprim, respectively. Resistance to mercury was carried by 33% of the plasmids, but none conferred significant resistance to silver or to any of three disinfectants tested. The potential virulence genes iutA (aerobactin system) and traT (serum survival) were carried by 21% and 36% of the plasmids, respectively. The 33 conjugative plasmids belonged to five different incompatibility groups, FIB, B/O, I1, K/B, and P (42%, 33%, 9%, 3% and 3%, respectively), with 3 plasmids being unassigned, and to 19 similarity groups on the basis of their restriction profiles. The sequences flanking sul2 were diverse and suggested more than one mechanism of genetic mobility. The five nonconjugative plasmids were all related to p9123 (pBP1), which was previously found to confer a fitness advantage on its host. We propose that the persistence of sul2, despite the reduced rate of prescription of sulfonamides, is due to a combination of coselection by antibiotics still in common use, a lack of a selective disadvantage in sul2 carriage, and the genetic mobility of sul2.

The rag locus of Porphyromonas gingivalis contributes to virulence in a murine model of soft tissue destruction.

The rag locus of Porphyromonas gingivalis encodes a putative TonB-dependent outer membrane receptor, RagA, and a 55-kDa immunodominant antigen, RagB. Inactivation of either ragA or ragB prevented expression of both RagA and RagB. Both the ragA and ragB mutants were significantly less virulent than wild-type strains in a murine model of infection.

Hypermutable bacteria isolated from humans--a critical analysis.

Hypermutable bacteria of several species have been described among isolates recovered from humans over the last decade. Interpretation of the literature in this area is complicated by diversity in the determination and definition of hypermutability, and this review outlines the different methods used. Inactivation of the mismatch repair gene mutS is often implicated in the mutator phenotype; the reported effect of mutS inactivation on mutation frequency varies widely between species, from under 10-fold to nearly 1,000-fold, but also varies among different reports on the same species. Particularly high proportions of mutators have been reported among Pseudomonas aeruginosa and other species in the cystic fibrosis lung, epidemic serogroup A Neisseria meningitidis, and Helicobacter pylori. Aspects of the biology of these infections that could be relevant to hypermutability are discussed, and some future directions that may increase our understanding of mutators among bacteria isolated from humans are considered.

Mutators among CTX-M beta-lactamase-producing Escherichia coli and risk for the emergence of fosfomycin resistance.

OBJECTIVES: Fosfomycin is a possible oral treatment for lower urinary tract infections caused by Escherichia coli with CTX-M extended-spectrum beta-lactamases but is vulnerable to mutational resistance. Hypermutability among natural E. coli populations might facilitate the emergence of resistance to fosfomycin. We therefore examined the prevalence of mutators amongst urinary isolates of E. coli producing CTX-M beta-lactamases. METHODS: Urinary E. coli isolates with CTX-M beta-lactamases (n = 220) were screened for resistance to both rifampicin and fosfomycin, as well as a mutator phenotype, by rifampicin and fosfomycin disc assays. Mutation frequencies for 10 isolates, identified as mutators by the initial disc screen, were determined in triplicate on agar with rifampicin or fosfomycin at 4x MIC and with fosfomycin or nitrofurantoin at 256 mg/L. RESULTS: The disc screen identified 10 likely mutators and quantitative tests indicated that 9 of these had mutation frequencies of 8.0 x 10(-6)-1.5 x 10(-4) for fosfomycin and 0.1-2.3 x 10(-6) for rifampicin. These mutators were diverse in terms of PFGE type and 4 of the 10 were confirmed as strong mutators with rifampicin and fosfomycin. Only the strongest mutator isolate and hypermutable MutS(-) control strain consistently gave single-step mutants resistant to 256 mg/L fosfomycin. No nitrofurantoin-resistant mutants were selected from any isolate, although they could be selected from the hypermutable MutS(-) control strain. CONCLUSIONS: Mutator phenotypes were found among E. coli expressing CTX-M beta-lactamases and were independent of strain type. These had an increased propensity to fosfomycin resistance.

Effect of subinhibitory concentrations of antibiotics on mutation frequency in Streptococcus pneumoniae.

OBJECTIVES: To investigate the effect of subinhibitory concentrations of ciprofloxacin, streptomycin, trimethoprim, ampicillin and erythromycin on mutation frequency in Streptococcus pneumoniae. METHODS: Frequency of mutation to rifampicin resistance was determined in three clinical isolates grown with or without antibiotic treatment. dinB was analysed using PCR and DNA sequence determination. RESULTS: Subinhibitory levels of ciprofloxacin and streptomycin increased the frequency of mutation to rifampicin resistance between 2- and 5-fold for all three isolates, which is comparable to the increase seen in mismatch repair mutants of this species. These increases appeared not to be dependent on the function of the error-prone DNA polymerase encoded by dinB, since one of the isolates was a naturally occurring deletion mutant for this gene. Trimethoprim increased the mutation frequency for two isolates, but not the dinB mutant; ampicillin and erythromycin had no significant effect on mutation frequencies for any isolate. CONCLUSIONS: Exposure to quinolones and aminoglycosides at subinhibitory concentrations may result in increased mutability in pneumococci, as well as selecting for resistance per se.

Resistance among Escherichia coli to sulphonamides and other antimicrobials now little used in man.

OBJECTIVES: We investigated whether sulphonamide resistance in Escherichia coli remained prevalent in 2004, 9 years since the formal introduction of a UK prescribing restriction on co-trimoxazole. Resistance to other agents no longer in common use was also examined. METHODS: Consecutive urinary E. coli isolates were obtained at the diagnostic microbiology laboratory of the Royal London Hospital from January to March 2004. The presence of the sulphonamide resistance genes, sul1, sul2 and sul3, and the class I integrase gene, int1, were determined by PCR. RESULTS: Of the 391 E. coli isolates recovered in 2004, 45.5% were sulphonamide-resistant compared with 46.0% in 1999 and 39.7% in 1991. The sul2 gene remained the most prevalent sulphonamide resistance determinant, present in 81% of resistant isolates in 2004 compared with 79% and 67% in 1999 and 1991, respectively; 28% of resistant isolates carried both sul1 and sul2 genes; sul3 was not found. Resistance to streptomycin also remained common, whereas resistance to chloramphenicol and kanamycin had decreased since 1999. CONCLUSION: Sulphonamide resistance in E. coli persists undiminished despite the prolonged withdrawal of this antibiotic in the UK; resistance to streptomycin also seems stable whilst that to chloramphenicol and kanamycin is declining.

Sequence diversity and antigenic variation at the rag locus of Porphyromonas gingivalis.

The rag locus of Porphyromonas gingivalis W50 encodes RagA, a predicted tonB-dependent receptor protein, and RagB, a lipoprotein that constitutes an immunodominant outer membrane antigen. The low G+C content of the locus, an association with mobility elements, and an apparent restricted distribution in the species suggested that the locus had arisen by horizontal gene transfer. In the present study, we have demonstrated that there are four divergent alleles of the rag locus. The original rag allele found in W50 was renamed rag-1, while three novel alleles, rag-2 to rag-4, were found in isolates lacking rag-1. The three novel alleles encoded variants of RagA with 63 to 71% amino acid identity to RagA1 and each other and variants of RagB with 43 to 56% amino acid identity. The RagA/B proteins have homology to numerous Bacteroides proteins, including SusC/D, implicated in polysaccharide uptake. Monoclonal and polyclonal antibodies raised against RagB1 of P. gingivalis W50 did not cross-react with proteins from isolates carrying different alleles. In a laboratory collection of 168 isolates, 26% carried rag-1, 36% carried rag-2, 25% carried rag-3, and 14% carried rag-4 (including the type strain, ATCC 33277). Restriction profiles of the locus in different isolates demonstrated polymorphism within each allele, some of which is accounted for by the presence or absence of insertion sequence elements. By reference to a previously published study on virulence in a mouse model (M. L. Laine and A. J. van Winkelhoff, Oral Microbiol. Immunol. 13:322-325, 1998), isolates that caused serious disease in mice were significantly more likely to carry rag-1 than other rag alleles.

Enhancement of host fitness by the sul2-coding plasmid p9123 in the absence of selective pressure.

OBJECTIVES: Despite a 97% reduction in clinical sulphonamide usage, the prevalence of sulphonamide resistance among Escherichia coli has remained constant in the UK. Genetic linkage of sulphonamide resistance to other resistances is thought important for this maintenance, but the finding also implies that sulphonamide resistance exerts little fitness cost. To test this hypothesis, we examined the fitness impact of four naturally occurring sul2-coding plasmids upon their hosts. METHODS: The fitness impact of the plasmids upon E. coli was determined by pairwise growth competition in a minimal medium. The DNA sequence of plasmid p9123 was obtained by primer walking and PCR. RESULTS: Three of the four sul2-coding plasmids studied imposed fitness costs on their hosts. The fourth plasmid, a 6.2 kb resistance element carrying sul2, strA and strB designated p9123, conferred a 4% fitness advantage upon its original clinical host and also on E. coli K12 JM109. The complete sequence of p9123 revealed eight open reading frames, including five of unknown function. There was no obvious gene to which the fitness advantage might be attributed. CONCLUSIONS: The novel finding that p9123 can improve host fitness may explain why this plasmid and its close relatives are so widespread among enteric bacteria. In addition to other factors such as co-selection of sulphonamide resistance by other agents, the fitness advantage conferred by plasmids such as p9123 may have contributed to the maintenance of sulphonamide resistance in the UK in the absence of clinical selection pressure. These data indicate that once antibiotic resistance has been established on mobile genetic elements, it may be difficult to eliminate.

Klebsiella pneumoniae isolate from South Africa with multiple TEM, SHV and AmpC beta-lactamases.

Klebsiella pneumoniae 2207, from Durban, was resistant to cefoxitin and beta-lactamase inhibitor combinations as well as oxyimino-aminothiazolyl cephalosporins. Beta-lactamases with isoelectric points of 5.4, 5.6, 7.6, 8.2 and 8.4 were found. DNA hybridisation identified two BamHI and three HindIII fragments carrying blaTEM, and two SalI fragments carrying blaSHV. At least two genes encoded TEM-1 enzyme; one blaSHV copy encoded SHV-5 but the other determined SHV-23, a novel SHV-5 variant with conservative amino-acid substitutions far from the catalytic site. The pI 8.4 activity was an AmpC-type enzyme. Determinants of the pI 5.6 and 7.6 activities were not identified.

Variable susceptibility to piperacillin/tazobactam amongst Klebsiella spp. with extended-spectrum beta-lactamases.

MICs of piperacillin/tazobactam are conventionally determined by varying the concentration of piperacillin in the presence of a fixed 4 mg/L tazobactam. When tested in this way, the MIC distribution for Klebsiella isolates with extended-spectrum beta-lactamases (ESBLs) is strongly bimodal, such that many producers are inhibited at 16 + 4 mg/L whilst others require MICs of > or =512 + 4 mg/L. When, however, piperacillin/tazobactam was tested as a fixed 8:1 ratio, the MIC distribution became unimodal. If clavulanate 4 mg/L was combined with piperacillin, a unimodal MIC distribution was seen for ESBL-producing Klebsiella spp. but a bimodal distribution arose if the clavulanate concentration was reduced to 0.25 mg/L. These data for alternative combinations suggested that the bimodal MIC distribution seen for piperacillin + tazobactam 4 mg/L was a titration effect, not a reflection of some ESBLs being resistant to tazobactam. Even within single strains, as defined by serotype and DNA fingerprints, there was considerable variation in susceptibility to piperacillin + tazobactam 4 mg/L, with some representatives highly susceptible and others highly resistant. Some of the more resistant representatives produced more of their ESBL, or had a greater number of beta-lactamase types, but these associations were not universal. Elevated resistance to piperacillin + tazobactam was not associated with porin change in any ESBL producer examined, but has been found by others.

Evaluation of semiautomated multiplex PCR assay for determination of Streptococcus pneumoniae serotypes and serogroups.

A semiautomated method for the determination of five serotypes and three serogroups in Streptococcus pneumoniae was developed. Primers specific for serotypes 1, 3, 14, 19F, and 23F and serogroups 6, 19, and 23 were combined in three multiplex PCRs. Products were separated by capillary electrophoresis with a 7-min run time, and a serotype or serogroup was assigned on the basis of fragment size. The method was used to test 93 clinical isolates, and all isolates of the serotypes concerned were correctly detected. The strategy would allow the detection of multiple serotypes in a single sample. Detection of additional serotypes could be included as capsule locus sequences become available.

Sulfonamide resistance in Haemophilus influenzae mediated by acquisition of sul2 or a short insertion in chromosomal folP.

Determinants of sulfonamide resistance were investigated in clinical isolates of Haemophilus influenzae from the United Kingdom and Kenya. The mechanism of sulfonamide resistance in H. influenzae has not previously been reported. Eight isolates requiring at least 1,024 microg of sulfamethoxazole per ml for inhibition carried the sul2 gene, a common mediator of acquired sulfonamide resistance in enteric bacteria. In other isolates with similarly high levels of resistance, the chromosomal gene encoding dihydropteroate synthase, folP, was found to carry an insertion of 15 bp together with other missense mutations relative to folP of H. influenzae strain Rd RM118 (MIC, 8 microg/ml); the folP sequence was identical in all seven such isolates investigated, although they represented three different strains by restriction pattern analysis. The 15-bp insertion was absent in isolates inhibited by sulfamethoxazole at 2 to 64 microg/ml (although these exhibited considerable divergence in folP sequence) and in highly resistant isolates carrying sul2. Transformation with a 599-bp fragment of folP containing the insertion but no other differences conferred high-level resistance on a recipient strain, confirming the role of the insertion. Other amino acid substitutions in dihydropteroate synthase may modulate the level of sulfonamide inhibition in susceptible isolates and those with more moderate levels of resistance. The two mechanisms of resistance, mediated by sul2 and modified folP, were detected in isolates from both the United Kingdom and Kenya.