In Vivo Selection of Pan-Drug Resistant Acinetobacter baumannii during Antibiotic Treatment

Purpose

Colistin resistance in Acinetobacter baumannii (A. baumannii) is mediated by a complete loss of lipopolysaccharide production via mutations in lpxA, lpxC, and lpxD gene or lipid A modifications via mutations in the pmrA and pmrB genes. However, the exact mechanism of therapy-induced colistin resistance in A. baumannii is not well understood.

Materials and Methods

We investigated the genotypic and phenotypic changes that underlie pan-drug resistance mechanisms by determining differences between the alterations in extensively drug-resistant (XDR) A. baumannii (AB001 and AB002) isolates and a pan-drug resistant (PDR) counterpart (AB003) recovered from one patient before and after antibiotic treatment, respectively.

Results

All three clinical isolates shared an identical sequence type (ST138), belonging to the global epidemic clone, clonal complex 92, and all produced OXA-23 carbapenemase. The PDR AB003 showed two genetic differences, acquisition of armA gene and an amino acid substitution (Glu229Asp) in pmrB gene, relative to XDR isolates. No mutations were detected in the pmrA, pmrC, lpxA, lpxC, or lpxD genes in all three isolates. In matrix-assisted laser desorption ionization-time of flight analysis, the three isolates commonly showed two major peaks at 1728 m/z and 1912 m/z, but peaks at 2034 m/z, 2157 m/z, 2261 m/z, and 2384 m/z were detected only in the PDR A. baumannii AB003 isolate.

Conclusion

Our results show that changes in lipid A structure via a mutation in the pmrB gene and acquisition of armA gene might confer resistance to colistin and aminoglycosides to XDR A. baumannii strains, resulting in appearance of a PDR A. baumannii strain of ST138.     

Endogenous Pro- and Anti-Inflammatory Cytokines Differentially Regulate an In Vivo Humoral Response to Streptococcus pneumoniae

Proinflammatory cytokines play a critical role in innate host defense against extracellular bacteria. However, little is known regarding the effects of these cytokines on the adaptive humoral response. Mice injected with a neutralizing anti-tumor necrosis factor alpha (TNF-alpha) monoclonal antibody (MAb) at the time of primary immunization with intact Streptococcus pneumoniae (strain R36A) showed a substantial reduction in both the primary immunoglobulin G (IgG) response specific for the cell wall protein, pneumococcal surface protein A (PspA), as well as in the development of PspA-specific memory. In contrast, anti-TNF-alpha MAb injected only at the time of secondary immunization with R36A failed to alter the boosted anti-PspA response. TNF-alpha was required only within the first 48 to 72 h after primary immunization with R36A and was induced both by non-B and non-T cells and by lymphoid cells, within 2 to 6 h after immunization, with levels returning to normal by 24 h. Thus, the early innate release of TNF-alpha was critical for optimal stimulation of the subsequent adaptive humoral response to R36A. Additional proinflammatory (interleukin 1 [IL-1], IL-6, IL-12, and gamma interferon [IFN-gamma]) as well as anti-inflammatory (IL-4 and IL-10) cytokines were also transiently induced. Mice genetically deficient in IL-6, IFN-gamma, or IL-12 also showed a reduced IgG anti-PspA response of all IgG isotypes. In contrast, IL-4(-/-) and IL-10(-/-) mice immunized with R36A showed a significant elevation in the IgG anti-PspA response, except that there was decreased IgG1 in IL-4(-/-) mice. In this regard, a marked enhancement in the induction of proinflammatory cytokines was observed in the absence of IL-10, relative to controls. Ig isotype titers specific for the phosphorycholine determinant of C-polysaccharide were similarly regulated, but to a much more modest degree. These data suggest that proinflammatory and anti-inflammatory cytokines differentially regulate an in vivo protein- and polysaccharide-specific Ig response to an extracellular bacteria.     

Characterization of erythromycin-resistant Streptococcus pneumoniae in Korea, and In Vitro activity of telithromycin against erythromycin-resistant Streptococcus pneumoniae

To characterize the phenotypes and genotypes of erythromycin-resistant clinical isolates of Streptococcus pneumoniae in Korea and to evaluate the in vitro activity of telithromycin against these erythromycin-resistant isolates, we tested a total of 676 isolates of S. pneumoniae collected from 1997 to 2002 in a tertiary hospital in Seoul, Republic of Korea. MICs for erythromycin and telithromycin were determined by the agar dilution method. The macrolide resistance phenotypes of erythromycin-resistant isolates were determined by the erythromycin- clindamycin-rokitamycin triple disk (ECRTD) and MIC induction tests, whereas their macrolide resistance genotypes were determined by PCR for the erm(B), erm(A), subclass erm(TR), and mef genes. To discriminate between mef(A) and mef(E), PCR-restriction fragment length polymorphism (RFLP) analyses were performed. Of the 676 S. pneumoniae isolates, 459 (67.9%) were resistant to erythromycin. Of the 459 erythromycin-resistant isolates, 343 (74.7%) were assigned to the cMLS phenotype, 48 (10.4%) to the iMcLS phenotype, 4 (0.9%) to the iMLS phenotype, and 64 (14.0%) to the M phenotype. The erm(B) gene was detected in 251 (54.6%) isolates, the mef gene was detected in 64 (14.0%), and both the erm(B) and mef genes were detected in 144 (31.4%) isolates. All of the mef genes detected were identified as mef(E). Of the 459 erythromycin- resistant isolates, all but one were susceptible to telithromycin. The MIC(50)/MIC(90) to telithromycin of isolates carrying erm(B), mef(E), and both genes was 0.06/0.5 microg/ml, 0.03/0.125 microg/ml, and 0.5/1.0 microg/ml, respectively. Although the MICs of telithromycin for the erythromycin-resistant isolates varied according to genotype, telithromycin was very active against these erythromycin-resistant S. pneumoniae.     

In vitro activity of pristinamycin against Streptococcus pneumoniae

The activity of the pristinamycin was investigated using disk diffusion agar or ATB PNEUMO system and MIC determination using reference liquid medium method (NCCLS) on 749 S. pneumoniae strains isolated in Aquitaine in 1999. We have realized the killing curves against 10 isolates selected from erythromycin-susceptible and resistant S. pneumoniae strains. All the strains tested by ATB PNEUMO system were susceptible to pristinamycin, using disk diffusion agar, 6.8% of strains were intermediate or resistant. However the MIC's of pristinamycin determined by liquid dilution method against these strains were < 1 mg/L. These data suggest that the zone of inhibition around the disk was not correlated with MIC for erythromycin pneumococci and MIC testing must be performed. The results of killing curves showed a very good and rapid bactericidal activity of pristinamycin within two hours for concentration equal to 4 x MIC and four hours for 2 x MIC.     

The Two Variants of the Streptococcus pneumoniae Pilus 1 RrgA Adhesin Retain the Same Function and Elicit Cross-Protection In Vivo

Thirty percent of Streptococcus pneumoniae isolates contain pilus islet 1, coding for a pilus composed of the backbone subunit RrgB and two ancillary proteins, RrgA and RrgC. RrgA is the major determinant of in vitro adhesion associated with pilus 1, is protective in vivo in mouse models, and exists in two variants (clades I and II). Mapping of the sequence variability onto the RrgA structure predicted from X-ray data showed that the diversity was restricted to the “head” of the protein, which contains the putative binding domains, whereas the elongated “stalk” was mostly conserved. To investigate whether this variability could influence the adhesive capacity of RrgA and to map the regions important for binding, two full-length protein variants and three recombinant RrgA portions were tested for adhesion to lung epithelial cells and to purified extracellular matrix (ECM) components. The two RrgA variants displayed similar binding abilities, whereas none of the recombinant fragments adhered at levels comparable to those of the full-length protein, suggesting that proper folding and structural arrangement are crucial to retain protein functionality. Furthermore, the two RrgA variants were shown to be cross-reactive in vitro and cross-protective in vivo in a murine model of passive immunization. Taken together, these data indicate that the region implicated in adhesion and the functional epitopes responsible for the protective ability of RrgA may be conserved and that the considerable level of variation found within the “head” domain of RrgA may have been generated by immunologic pressure without impairing the functional integrity of the pilus.Streptococcus pneumoniae is a main determinant of respiratory tract infections, such as otitis media, sinusitis, and community-acquired pneumonia, and is also responsible for invasive diseases, such as bacteremic pneumonia and meningitis (16, 17, 30, 43, 46, 50, 55). Nonetheless, pneumococci are normal components of the human commensal flora, asymptomatically colonizing the upper respiratory tracts of both children and healthy adults. Colonization is commonly followed by horizontal transmission of S. pneumoniae, leading to its spread within the community (4, 19, 33). Current glycoconjugate vaccines are efficacious against invasive disease caused by serotypes included in the vaccines; however, their potential to prevent carriage and related mucosal diseases, such as otitis media, is not optimal (10, 12, 23, 29, 34, 48). Furthermore, the partial geographic coverage and the phenomenon of serotype replacement associated with the introduction of the 7-valent conjugate vaccine limit to some extent its long-term effectiveness (11, 20, 31, 37). For these reasons, current research is focused on the identification of protein vaccine candidates able to elicit serotype-independent protection against S. pneumoniae infection. In this context, colonization could represent a critical point of intervention, and bacterial components involved in these mechanisms should be studied in order to determine their value as vaccine candidates.Adhesion of bacteria to the mucosa is considered an essential early step in the colonization process. The ability of S. pneumoniae to adhere to epithelial cells has been ascribed to a number of surface-exposed proteins, including PspC, PsaA, PsrP, PfbB, NanA, PavA, and pili (3, 21, 41, 42, 45, 49, 54).Pili were recently discovered in many Gram-positive pathogens, and although their biological function has not been fully elucidated, their presence has been mostly related to bacterial adhesion, biofilm formation, and translocation of epithelial barriers (1, 13, 35, 47). These structures are composed of subunits covalently linked by means of intermolecular isopeptide bonds (32, 36, 51-53). Furthermore, intramolecular isopeptide bonds have been found in most pilus subunits characterized to date (9, 26-28). These bonds may play a critical role in maintaining pilus integrity in the face of severe mechanical and chemical stress while bound to host cells and thus may provide a functional mode of stabilization for cell surface proteins involved in host pathogenesis.In S. pneumoniae, pili (pilus 1 and pilus 2) are encoded by two genomic islets (pilus islet 1 [PI-1] and PI-2) that are not present in all pneumococcal clinical isolates. A number of molecular epidemiological studies have highlighted the presence of PI-1 as a clonal property of S. pneumoniae isolates and have defined, based on sequence analysis, the classification of PI-1 into three major clades (2, 5, 7, 25, 38, 39). Mutants lacking PI-1 are impaired in adhesion to cultured epithelial cells in vitro and are less virulent in murine models of colonization, pneumonia, and bacteremia (6, 41). Interestingly, pilus 1 expression is known to increase host inflammatory responses that might disrupt the mucosal barrier and facilitate subsequent invasion by the bacteria (6).Pneumococcal pilus 1 is composed of three subunits (RrgA, RrgB, and RrgC); RrgB is the backbone component, and RrgA is the major ancillary protein, localized at the pilus tip and responsible for the adhesion properties of the pilus, whereas RrgC is the minor ancillary protein, likely located at the pilus base (21, 22, 41). In terms of sequence variability, RrgB is classified into three variants and RrgC is conserved, whereas RrgA exists in two major variants (clades I and II) (38). The recombinant form of RrgA clade I adheres in vitro to cultured A549 lung epithelial cells, as well as to purified extracellular matrix (ECM) components (collagen I, fibronectin, and laminin) (21, 41). In addition, RrgA, along with the other two pilus 1 components, is able to elicit protection from lethal challenge with the homologous strain in mouse models of active and passive immunization (18).In this work, we investigated whether the differences between the two variants had an effect on the biochemical characteristics, biological function, and immunological properties of the molecule. We found that (i) sequence variability was restricted to the “head” domain of RrgA, containing the putative adhesive motifs; (ii) the two RrgA variants were resistant to proteolytic cleavage, and this feature was dependent on the presence of intramolecular isopeptide bonds; (iii) the two variants were able to adhere to epithelial cells and ECM components at comparable levels, whereas a mutant (Asp444Ala) in the RGD tripeptide showed reduced binding; (iv) none of the individual fragments encompassing the N-terminal (NT), central (CP), and C-terminal (CT) portions of RrgA was able to maintain adhesive capacity; (v) antibodies against these fragments revealed the N terminus to be less accessible than the remaining portion of the molecule on the native pilus; and, finally, (vi) antibodies raised against each of the two RrgA variants were cross-reactive and cross-protective in murine passive-immunization studies.     

Virulence, immunity, and vaccine related to Streptococcus pneumoniae

The pathogenesis of bacterial infection involves a series of interactions between the virulence determinants of the microorganisms and the immunity of the host. Studies on the molecular structure and immunological properties of pneumococcal virulence factors have provided general knowledge for the chemical basis of immunogenicity and prevention of bacterial infection. Antibody responses to PS and protein antigens can be greatly affected by their physicochemical properties, e.g., molecular size, specific determinants, conformation, etc. Characterization of group 19 pneumolysins and cloning of their ply genes were studied to examine the relationship of ply to virulence. Group 19 pneumococci all contained ply; the disease-isolated types of 19F and 19A appeared to show a higher specific hemolytic activity and yield than the nonpathogenic types, 19B and 19C. Genomic DNA that contained the ply gene from group 19 strains were analyzed by the polymerase chain reaction (PCR). Type 2 oligonucleotide primers recognized and initiated synthesis of an identical 1.5 kb DNA fragment in types 2, 19F, 19A, 19B, and 19C. Their sizes of restriction DNA fragments were also found to be homologous. Thus, group 19 ply genes showed remarkably similar characteristics. A difficult problem in the development of vaccines against bacterial diseases is the poor immune response of young children to purified PSs. The efficacy of pneumococcal vaccine might be improved by supplementation with inactivated pneumolysin in the form of a PS-protein conjugate.     

肺炎链球菌体内启动子诱捕文库的构建与初步分析

构建肺炎链球菌体内启动子诱捕文库(promoter-trap library),用于筛选肺炎链球菌体内诱导的基因。以穿梭质粒pEVP3为骨架,将无启动子的galU基因作为体内报告基因定向克隆到pEVP3上,与无启动子的体外报告基因lacZ基因融合,构建用于筛选体内诱导基因的载体pEVP3-galU;再把肺炎链球菌基因组DNA的随机酶切片段(200~500bp)克隆到此载体galU基因上游的Bgl II位点,构建了启动子诱捕文库,并转化肺炎链球菌galU缺陷菌株,获得相应的菌株库。文库大约覆盖基因组全长的5倍,插入率达到90%以上,保持了较高的复杂性。对得到的约450 000个肺炎链球菌转化子进行体内、外初步分析,那些从小鼠体内分离出的细菌,并在X-gal平板上为白色的菌落表明galU报告基因上游含仅在体内表达的启动子,提示所构建的启动子诱捕文库可用于筛选肺炎链球菌的体内诱导基因。     

In Vitro Susceptibility to Gemifloxacin and Trovafloxacin of Streptococcus pneumoniae Strains Exhibiting Decreased Susceptibility to Ciprofloxacin

 The in vitro susceptibility to trovafloxacin and gemifloxacin of Streptococcus pneumoniae strains exhibiting decreased susceptibility to ciprofloxacin (MIC ≥2 μg/ml; 30 strains with intermediate resistance [MIC 2 μg/ml] and 43 strains with complete resistance [MIC ≥4 μg/ml]) was determined. Seventy-three strains collected in a surveillance study carried out from May 1996 to April 1997 in Spain (prior to commercialisation of trovafloxacin and gemifloxacin) from patients with respiratory tract infections were tested. The antibacterial activity of gemifloxacin was affected to a lesser extent than that of trovafloxacin by the increase in the MIC of ciprofloxacin, with gemifloxacin showing significantly (P≤0.001) better antibacterial activity than trovafloxacin in all ciprofloxacin MIC categories (MIC50/MIC90 values of 0.015/0.03, 0.015/0.06, 0.03/0.06 and 0.12/0.25 μg/ml for gemifloxacin vs 0.12/0.12, 0.12/1, 0.25/0.5 and 2/4 μg/ml for trovafloxacin in the 2, 4, 8 and ≥16 μg/ml ciprofloxacin MIC categories, respectively). Nine (12.3%) of these 73 strains exhibited decreased susceptibility to trovafloxacin (≥2 μg/ml), whereas all strains were inhibited by 0.25 μg/ml of gemifloxacin.     

In Vitro Plaque-Forming Cell Responses Induced by Streptococcus pneumoniae in Humans

When peripheral blood lymphocytes (PBL) were stimulated in vitro with the rough form of type 2 Streptococcus pneumoniae R36a, the resulting plaque-forming cells (PFC) did not produce antibodies directed against phosphorylcholine, a major antigenic determinant of the cell wall C-polysaccharide. Instead, R36a stimulated polyclonal PFC in PBL and splenic lymphocytes. We compared the polyclonal responses stimulated by R36a with those induced by two well-characterized polyclonal activators (PA), Staphylococcus aureus Cowan I and pokeweed mitogen (PWM). We found that R36a was a poor mitogen for PBL, whereas the other two PA were potent mitogens; that the predominant isotype produced in response to all three PA was IgM; that adherent cells strongly inhibited the polyclonal PFC response to both R36a and Staph. aureus but not PWM; and that T cells were necessary for induction of polyclonal antibody-secreting cells by all three stimuli.     

Proteomic response of Streptococcus pneumoniae to iron limitation

Iron is an essential trace element and involved in various key metabolic pathways in bacterial lifestyle. Within the human host, iron is extremely limited. Hence, the ability of bacteria to acquire iron from the environment is critical for a successful infection. Streptococcus pneumoniae (the pneumococcus) is a human pathobiont colonizing symptomless the human respiratory tract, but can also cause various local and invasive infections. To survive and proliferate pneumococci have therefore to adapt their metabolism and virulence factor repertoire to different host compartments. In this study, the response of S. pneumoniae to iron limitation as infection-relevant condition was investigated on the proteome level. The iron limitation was induced by application of the iron chelator 2,2′-bipyridine (BIP) in two different media mimicking different physiological traits. Under these conditions, the influence of the initial iron concentration on pneumococcal protein expression in response to limited iron availability was analyzed. Interestingly, one major difference between these two iron limitation experiments is the regulation of proteins involved in pneumococcal pathogenesis. In iron-poor medium several proteins of this group were downregulated whereas these proteins are upregulated in iron-rich medium. However, iron limitation in both environments led to a strong upregulation of the iron uptake protein PiuA and the significant downregulation of the non-heme iron-containing ferritin Dpr. Based on the results, it is shown that the pneumococcal proteome response to iron limitation is strongly dependent on the initial iron concentration in the medium or the environment.     

Adherence of Streptococcus pneumoniae to immobilized fibronectin.

Adherence to extracellular matrix proteins, such as fibronectin, affords pathogens with a mechanism to invade injured epithelia. Streptococcus pneumoniae was found to adhere to immobilized fibronectin more avidly than other streptococci and staphylococci do. Binding was dose, time, and temperature dependent. Trypsin treatment of the bacteria resulted in decreased binding, suggesting that the bacterial adhesive component was a protein. Fragments of fibronectin generated by proteolysis or by expression of recombinant gene segments were compared for the ability to bind pneumococci and to compete against bacterial binding to immobilized fibronectin. Fragments from the carboxy-terminal heparin binding domain were consistently active, suggesting that this region contains the pneumococcal binding site, a region distinct from that supporting the attachment of most other bacteria.     

Management of Infections Due to Antibiotic-Resistant Streptococcus pneumoniae

Antibiotic-resistant strains of Streptococcus pneumoniae are becoming more prevalent throughout the world; this has resulted in modifications of treatment approaches. Management of bacterial meningitis has the greatest consensus. Strategies for treating other systemic infections such as pneumonia, bacteremia, and musculoskeletal infections are evolving, in part related to the availability of new antibiotics which are active in vitro against isolates resistant to penicillin and the extended-spectrum cephalosporins. However, there are currently very limited data related to the clinical efficacy of these new agents. The studies upon which current recommendations are based are reviewed. Otitis media represents the single most common infection due to S. pneumoniae. Recommendations for treatment of acute otitis media due to drug-resistant strains and the rationale for these recommendations are discussed.     

Molecular Epidemiology of Streptococcus pneumoniae Serogroup 6 Isolates from Fijian Children,Including Newly Identified Serotypes 6C and 6D

Multilocus sequence typing (MLST) was applied to all unique serotype 6C and 6D isolates and a random selection of serotype 6B and 6A isolates from nasopharyngeal swabs from Fijian children enrolled in a recent vaccine trial. The results suggest that Fijian serotype 6D has arisen independently from both serotypes 6A/C and 6B.Infection with Streptococcus pneumoniae is a leading cause of death in children worldwide (15, 19, 25). S. pneumoniae comprises 48 capsular serogroups containing more than 90 serotypes. Serogroup 6 classically consisted of serotypes 6A and 6B. The 7-valent conjugate vaccine (Prevnar; PCV7) includes the 6B antigen and confers some cross-protection against serotype 6A but not 6C (20, 22). Serotype 6C is important in carriage and invasive disease, and its prevalence has increased following widespread use of PCV7 (4, 5, 9, 10, 14, 16, 24).The existence of serotype 6D has been postulated (8) and was created experimentally (2), but naturally occurring isolates have not been identified (2, 8, 17) until recently, when we identified 14 naturally occurring serotype 6D isolates from nasopharyngeal swabs from Fijian children (11). Recently, two serotype 6D isolates have been found in Korean children (1).Initially, serotype 6C was postulated to have arisen from a single, possibly recent, evolutionary event in which serotype 6A wciN was replaced by serotype C wciN (21). Subsequent analyses, predominantly by multilocus sequence typing (MLST), have shown that 6C is genetically diverse, believed to be a consequence of multiple separate conversion events or a single event occurring sufficiently early in pneumococcal evolution (3, 4, 8, 9, 17).Serotype 6C is usually associated with clonal complexes (CCs) containing predominantly serotype 6A, and less commonly, 6B or non-serogroup-6 serotypes (3, 4, 9, 17). To date, MLST has been conducted on two serotype 6D isolates from Korea (ST282) (1), two from China (ST982 and ST4190), and one from Australia (ST4241) (http://spneumoniae.mlst.net). However, the molecular epidemiology of serotype 6D isolates is otherwise uncharacterized.In this study, we conducted MLST of serogroup 6 isolates to determine the genetic diversity and likely evolutionary origin of serotype 6C and 6D isolates.S. pneumoniae was isolated and identified as described elsewhere (18) from children aged 6 to 18 months participating in the Fiji Pneumococcal Project (FiPP) who had received 0, 1, 2, or 3 doses of PCV7 and 0 or 1 dose of 23-valent pneumococcal polysaccharide vaccine (PPV23) (23). Isolates were serotyped by a multiplex PCR-based reverse line blot (mPCR/RLB) assay and/or quellung reaction (before factor serum 6d was available), plus serogroup 6 serotype-specific PCR as previously described (11, 12).MLST was applied to all unique serotype 6C and 6D strains from the FiPP study (n = 52 and 24, respectively), of which 24 and 14 isolates, respectively, were included in our previous report (without ST results) (11). For comparison, we performed MLST on a subset of randomly selected serotype 6A (n = 16) and 6B (n = 17) isolates from the FiPP study.Fresh 18- to 24-h subcultures of S. pneumoniae isolates were suspended in nuclease-free water (Ambion), and genomic DNA was extracted using the DNeasy blood and tissue kit (Qiagen) per the manufacturer''s instructions. MLST was performed using primer pairs described by the Centers for Disease Control and Prevention (http://www.cdc.gov/ncidod/biotech/strep/alt-MLST-primers.htm) (aroE, recP, spi, xpt and ddl) or Enright and Spratt (6) (gdh and gki), except as described below.PCRs were conducted with 25-μl volumes containing approximately 5 ng of genomic DNA, 1 U of AmpliTaq DNA polymerase (Applied Biosystems), 1 × PCR buffer II (50 mM KCl, 10 mM Tris-HCl [pH 8.3]), 3.0 mM MgCl₂, 250 μM each deoxynucleoside triphosphate (dNTP), 0.5 μM forward primer, and 0.5 μM reverse primer (Sigma-Aldrich). PCR cycling conditions were a 5-min hold at 94°C, followed by 35 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s, and a final extension at 72°C for 5 min. Some isolates which produced no or small amounts of PCR product from spi (seven 6C and three 6D isolates) and/or recP (three 6C and one 6A isolate) were successfully amplified with primers described by Enright and Spratt (6) at an annealing temperature of 52°C in 4.5 mM MgCl₂.Amplicons were sequenced in both directions using capillary separation on the ABI 3730xl DNA analyzer with ABI BigDye Terminator labeling (version 3.1) (Australian Genome Research Facility) using the same primers as for amplification. Contiguous sequences were formed and edited using Sequencher 4.9 (Gene Codes Corporation).Allelic profiles and sequence types (STs) were obtained and compared to those of other isolates in the MLST database (http://spneumoniae.mlst.net). Relationships between STs were explored using eBURST version 3 software (Imperial College, London), which is available at the MLST website. For this study, a group was defined as two or more isolates which shared alleles at six of seven loci. Using this stringent definition, a group also defined a clonal complex (CC). Bootstrap analyses of the CC founder are also presented where appropriate.Generally, Fijian serogroup 6 isolates were highly clonal, with the predominant clone in each serotype representing >44% of the isolates. This is not surprising, given that all strains were isolated from children of similar ages over a relatively short period of time in a small, geographically isolated area.Serotype 6A isolates included STs 490 (7/16), 4778, 4779, 499, and 460 (Table ​(Table1).1). ST490 is predicted to be the CC founder (bootstrap value, 96%) and contains predominantly serotype 6A isolates in the MLST database. ST4778 and ST4779 are newly identified in this study and were not assigned to a CC. The only other ST499 isolate in the database (serotype 6A isolate from Finland) was also not assigned to a CC. ST460 is predicted to be a CC founder (bootstrap value, 88%). Most serotype 6A isolates (10/16) belonged to STs which contain only serotype 6A in the database (i.e., ST490, ST499, and ST460).

TABLE 1.

Distribution of STs and eBURST analysis for 109 serotype 6 isolates from Fijian children

In vitro antibacterial activity of beta-lactams and non-beta-lactams against Streptococcus pneumoniae isolates from Sydney, Australia

AIMS: This study was undertaken to determine the antimicrobial resistance patterns of strains of Streptococcus pneumoniae from Sydney, Australia, comparing penicillin-susceptible, -intermediate and -resistant isolates. METHODS: Non-duplicate cultures of S. pneumoniae were collected from 1 January to 31 December 2002 in the three penicillin-susceptibility categories. Minimum inhibitory concentrations (MICs) of 19 antibacterial agents were determined by agar dilution based on the National Committee for Clinical Laboratory Standards (NCCLS) methodology. Overall for 2002, 687 non-duplicate isolates were obtained, of which 190 (28%) were intermediate or resistant to penicillin. From this set, 183 isolates were selected for study: 88 (48%) in the penicillin-susceptible group (MIC or= 2.0 mg/L). RESULTS: Resistance to non-beta-lactams was more common in penicillin-intermediate or -resistant strains. Multidrug resistance (resistance to >or= 2 non-beta-lactams) was found in 3% of penicillin-susceptible, 52% of penicillin-intermediate and 87% of penicillin-resistant isolates. Erythromycin resistance was seen in 22% of the penicillin-susceptible strains but increased significantly to 60% and 89% in the penicillin-intermediate and resistant strains, respectively. Clindamycin, tetracycline and trimethoprim/sulfamethoxazole showed similar diminished activity in penicillin-intermediate and -resistant strains; 64, 84 and 91% of the penicillin-resistant isolates were resistant to clindamycin, tetracycline and to trimethoprim/sulfamethoxazole, respectively. Chloramphenicol resistance was comparatively low level except 19% of the penicillin-resistant strains were resistant. Ciprofloxacin MICs for 14 strains were raised (MICs 4-16 mg/L); three of these were penicillin-susceptible, one penicillin-intermediate and 10 penicillin-resistant. Only one isolate was resistant to moxifloxacin and to gatifloxacin. Resistance to rifampicin, vancomycin, oritavancin, or linezolid was not detected. Twenty-three isolates were intermediate and one resistant to quinupristin/dalfopristin - 22 of these were penicillin resistant. CONCLUSIONS: Streptococcus pneumoniae isolates from Sydney are commonly resistant to beta-lactams and available non-beta-lactam agents, especially if they are penicillin non-susceptible. Resistance to moxifloxacin and gatifloxacin is still rare, but some isolates were non-susceptible to quinupristin/dalfopristin. It is important to continue to survey resistance patterns to recognise emerging resistances which affect the selection of empirical antimicrobials to treat infections with S. pneumoniae.     

In Vivo Expression of Streptococcus pyogenes Immunogenic Proteins during Tibial Foreign Body Infection

Group A streptococcus (GAS) is an important human pathogen that causes a number of diseases with a wide range of severities. While all known strains of GAS are still sensitive to penicillin, there have been reports of antibiotic treatment failure in as many as 20% to 40% of cases. Biofilm formation has been implicated as a possible cause for these failures. A biofilm is a microbially derived, sessile community where cells grow attached to a surface or as a bacterial conglomerate and surrounded by a complex extracellular matrix. While the ability of group A streptococcus to form biofilms in the laboratory has been shown, there is a lack of understanding of the role of GAS biofilms during an infection. We hypothesized that during infections, GAS exhibits a biofilm phenotype, complete with unique protein expression. To test this hypothesis, a rabbit model of GAS osteomyelitis was developed. A rabbit was inoculated with GAS using an infected indwelling device. Following the infection, blood and tissue samples were collected. Histological samples of the infected tibia were prepared, and the formation of a biofilm in vivo was visualized using peptide nucleic acid fluorescent in situ hybridization (PNA-FISH) and confocal microscopy. In addition, Western blotting with convalescent rabbit serum detected cell wall proteins expressed in vitro under biofilm and planktonic growth conditions. Immunogenic proteins were then identified using matrix-assisted laser desorption ionization–time of flight tandem mass spectrometry (MALDI-TOF/TOF MS). These identities, along with the in vivo results, support the hypothesis that GAS forms biofilms during an infection. This unique phenotype should be taken into consideration when designing a vaccine or any other treatment for group A streptococcus infections.     

Design of a multiplex PCR for Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae and Chlamydophila pneumoniae to be used on sputum samples

A multiplex PCR (mPCR) was developed for simultaneous detection of specific genes for Streptococcus pneumoniae (lytA), Mycoplasma pneumoniae (P1), Chlamydophila pneumoniae (ompA), and Haemophilus influenzae (16S rRNA, with verification PCR for P6). When the protocol was tested on 257 bacterial strains belonging to 37 different species, no false negatives and only one false positive were noted. One Streptococcus mitis out of thirty was positive for lytA. In a pilot application study of 81 sputum samples from different patients with suspected lower respiratory tract infection (LRTI), mPCR identified S. pneumoniae in 25 samples, H. influenzae in 29, M. pneumoniae in 3, and C. pneumoniae in 1. All samples culture positive for S. pneumoniae (n=15) and H. influenzae (n=15) were mPCR positive for the same bacteria. In a pilot control study with nasopharyngeal swabs and aspirates from 10 healthy adults, both culture and mPCR were negative. No PCR inhibition was found in any of the mPCR-negative sputum or nasopharyngeal samples. Whether all samples identified as positive by mPCR are truly positive in an aetiological perspective regarding LRTI remains to be evaluated in a well-defined patient material. In conclusion, the mPCR appears to be a promising tool in the aetiological diagnostics of LRTI.     

Evolutionary barriers to quinolone resistance in Streptococcus pneumoniae

It is assumed that bacteria always pay a significant physiological price for the acquisition of resistance to antibiotics. To test whether this was the case for a strain of Streptococcus pneumoniae that develops resistance to fluoroquinolone antibiotics, we selected resistance to these agents in a wild-type strain and measured their fitness in comparative growth experiments. The relative growth rate of a mutant strain selected on ciprofloxacin (parC Serine 79 to Tyrosine) was compared with its susceptible isogenic parent and no significant deficit was found (relative fitness 1.15 95% C.I. +/- 0.2.). A double mutant, however, had a relative fitness of 0.81 (parC Serine 79 to Tyrosine gyrA Serine 81 to Tyrosine). Mutant strains selected on gemifloxacin had only a modest increase in minimum inhibitory concentration; thus, second-round mutants were competed with a first-round gyrA Serine 81 to Tyrosine or the susceptible isogenic parent. The growth rate of three double-mutant strains parC Serine 79 to Tyrosine gyrA Serine 81 to Phenylanine, parC Serine 79 to Tyrosine, and Asparagine 83 to Phenylalanine were similar to the isogenic susceptible parent 1.16 (95% C.I. +/- 0.17), 0.99 (95% C.I. +/- 0.05), and 0.95 (95% C.I. +/- 0.05), respectively. These data suggest that mutation in the parC and gyrA genes may, on some occasions, not be associated with a physiological deficit.