- Volume 62, Issue 9, 2013

Leptospira strains JICH 05 and INCIENSA 04 were isolated from hospitalized leptospirosis patients in the province of Puntarenas, Costa Rica. The isolates produced agglutination titres notably against members of serogroups Pyrogenes and Tarassovi, respectively, but appeared serologically unique in the cross agglutinin absorption test (CAAT). Therefore, JICH 05 and INCIENSA 04 were considered to represent two new serovars, designated Corredores and Costa Rica of the serogroups Pyrogenes and Tarassovi, respectively. Multilocus sequence genotyping revealed that both strain INCIENSA 04 and strain JICH 05 belong to Leptospira santarosai. These two new serovars are in addition to various other recently identified highly virulent serovars, including the new L. santarosai, serovar Arenal. Considering the fact that isolation and typing of leptospires from patients has only recently been introduced in Costa Rica, these findings suggest that various known and unknown virulent serovars of Leptospira are circulating in this country and probably beyond, thus posing a severe threat to public and probably veterinary health in the region.

In a rabbit model, we have previously reported evidence for a pathogenic role of streptococcal IgG Fc-binding proteins (IgGFcBP) in poststreptococcal glomerulonephritis (PSGN). These proteins, of the M protein family, were shown to trigger anti-IgG production and enhance renal deposition of IgG and/or immune complexes (ICs), with resulting activation of complement and cytokine cascades. In the present study, type M12/emm12, group A streptococci (GAS) were found often to bind artificial ICs, viz. peroxidase–anti-peroxidase rabbit IgG (PAP) or tetanus toxoid–anti-tetanus human IgG (TAT), rather than monomeric IgG. Animals injected with each of four IC binding clinical isolates (from patients with scarlet fever or PSGN) showed pronounced inflammatory and degenerative glomerular changes, morphologically similar to human PSGN, with membrane thickening and IgG and complement C3 deposition, as well as secretion of IL-6 and TNF-α by mesangial and endothelial cells. In contrast, non-binding strains (two from asymptomatic carriers and one from a PSGN case) failed to trigger any renal changes. Only the IC binding strains induced elevated titres of anti-IgG. Though the streptococcal binding component(s) has not been demonstrated, the selective binding of ICs by type M12/emm12 strains appears important for the well-known, marked nephritogenic potential of this GAS type.

Recent vaccination with pertussis vaccine can confound serological and oral fluid (OF) assays targeting anti-pertussis toxin (anti-PT) IgG antibodies as a marker of recent infection. This study sought to establish the minimum potentially confounding time period based on experimental data to assist interpretation from such samples submitted from UK subjects for pertussis diagnosis. Anti-PT IgG antibody response and decay were measured post-vaccination using a modified OF IgG antibody-capture ELISA (GACELISA). Data were obtained from 72 infants after the third acellular pertussis vaccine dose in the primary schedule (4 months of age) and from 119 children after the single dose at preschool age (3 years 4 months to 5 years 8 months of age). Specimens were taken at approximately 1 month intervals for 9 months post-primary immunization (third dose) and 13 months post-preschool booster (PSB). The modified GACELISA demonstrated a sensitivity of 52/56 (92.9 %: 95 % CI 82.7–98.0) and a specificity of 120/128 (93.8 %: 95 % CI 88.0–97.3) and showed good agreement with the National Reference Laboratory standard anti-PT IgG serum ELISA (rank correlation = 0.80) and the original OF assay (rank correlation = 0.79). Modelling of the decline in antibody titres showed a reduction of 54 % and 34 % for each doubling of time after day 14 for the post-third primary dose and post-PSB subjects, respectively. These data suggest that the minimum confounding time period is approximately 300 days for samples obtained post-primary immunization and at least 3 years for samples submitted from UK children following immunization with the PSB. These data will greatly assist the interpretation of single high diagnostic anti-PT IgG titres by allowing an estimate of the positive predictive value, when the number of days post-immunization and prevalence are known or assumed.

Strains of Porphyromonas gingivalis, a periodontopathic bacterium, are classified into six genotypic variants based on nucleotide sequence differences in the fimA gene encoding FimA. A PCR assay using primer sets specific for each genotype has demonstrated that the most predominant fimA genotype in periodontitis patients is type II, which is now commonly referred to as the periodontitis-associated fimA genotype of P. gingivalis. However, the potential for false type II fimA positives caused by cross-hybridization of type II fimA-specific primers with type Ib fimA has complicated the genotyping. A previous study developed new primers that specifically amplified only the DNA fragment of type II fimA. The aim of the present study was to assess the prevalence of P. gingivalis fimA genotypes in Korean adults and to reconfirm the relationship between type II fimA and periodontitis using the new primers. Among 412 Korean adults, P. gingivalis was detected in 97.5 % of patients and 57.8 % of healthy subjects. Type II fimA was the most widely distributed type among healthy and periodontitis subjects. Organisms with types II, Ib and IV fimA had a significant frequency of occurrence in periodontitis subjects. Statistical analysis, however, revealed that a more significant correlation was found between periodontitis and the occurrence of type Ib fimA.

Acinetobacter spp. are a diverse group of Gram-negative bacteria frequently implicated in nosocomial infections. Genotypic methods have been instrumental in studying Acinetobacter, but few offer high resolution, rapid turnaround time, technical ease and high inter-laboratory reproducibility, which has hampered understanding of disease incidence, transmission patterns and diversity within this genus. Here, we further evaluated multilocus PCR electrospray ionization/mass spectrometry (PCR/ESI-MS), a method that is simple and robust, and provides both species characterization and strain-level resolution of Acinetobacter spp. on a single platform. We examined 125 Acinetobacter isolates from 21 hospitals, laboratories and medical centres spanning four counties in Arizona, USA, using PCR/ESI-MS. We compared PCR/ESI-MS with an in-house amplified fragment length polymorphism (AFLP) genotyping scheme. PCR/ESI-MS demonstrated that Acinetobacter spp. from Arizonan hospitals had similar species and strain distributions to other US civilian hospitals. Furthermore, we showed that the PCR/ESI-MS and AFLP genotypes were highly congruent, with the former having the advantages of robust inter-laboratory reproducibility, rapid turnaround time and simple experimental set-up and data analysis. PCR/ESI-MS is an effective and high-throughput platform for strain typing of Acinetobacter baumannii and for identification of other Acinetobacter spp., including the emerging nosocomial pathogens Acinetobacter pittii and Acinetobacter nosocomialis.

We evaluated the performance of the Vitek MS for identification of Gram-positive cocci routinely isolated in clinical microbiology laboratories. With a total of 424 well-characterized isolates, the results of the Vitek MS were compared to those of conventional methods and 16S rRNA gene sequencing. The Vitek MS correctly identified 97.9 % of the isolates tested to species level. The Vitek MS correctly identified the species of 97.2 % of the staphylococci (95.9 % of coagulase-negative staphylococci), 97.8 % of the streptococci, and 100 % of the enterococci. For the identification of Gram-positive cocci isolates, the overall concordance rate between conventional identification and the Vitek MS was 94.5 %. The Vitek MS matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) system can be a reliable and rapid method for the identification of most relevant Gram-positive cocci. In addition, expanding the database of the Vitek MS, especially for coagulase-negative staphylococci, is needed to enhance the performance of the Vitek MS.

Prevotella intermedia, a major periodontopathogen, has been shown to be resistant to many antibiotics. In the present study, we examined the effect of the FDA-approved iron chelators deferoxamine (DFO) and deferasirox (DFRA) against planktonic and biofilm cells of P. intermedia in order to evaluate the possibility of using these iron chelators as alternative control agents against P. intermedia. DFRA showed strong antimicrobial activity (MIC and MBC values of 0.16 mg ml−1) against planktonic P. intermedia. At subMICs, DFRA partially inhibited the bacterial growth and considerably prolonged the bacterial doubling time. DFO was unable to completely inhibit the bacterial growth in the concentration range tested and was not bactericidal. Crystal violet binding assay for the assessment of biofilm formation by P. intermedia showed that DFRA significantly decreased the biofilm-forming activity as well as the biofilm formation, while DFO was less effective. DFRA was chosen for further study. In the ATP-bioluminescent assay, which reflects viable cell counts, subMICs of DFRA significantly decreased the bioactivity of biofilms in a concentration-dependent manner. Under the scanning electron microscope, P. intermedia cells in DFRA-treated biofilm were significantly elongated compared to those in untreated biofilm. Further experiments are necessary to show that iron chelators may be used as a therapeutic agent for periodontal disease.

Clonal dissemination of multidrug-resistant Pseudomonas aeruginosa (MDRPA) is a major concern worldwide. The aim of this study was to explore the mechanisms leading to the carbapenem resistance of an MDRPA clone. Isolates were obtained from a surgical wound, sputum, urine and a blood culture. Pulsed-field gel electrophoresis (PFGE) showed high genomic homogeneity of these isolates and confirmed the circulation of an endemic clone belonging to serotype O4. Outer membrane protein (OMP) bands were visualized by SDS-PAGE, meropenem accumulation was measured in a bioassay and integrons were detected by PCR. Efflux pumps were studied for several antimicrobial agents and synergic combinations thereof in the presence or absence of both carbonyl cyanide m-chlorophenylhydrazone (CCCP) and Phe-Arg-β-naphthylamide (PAβN) at final concentrations of 10 and 40 mg l−1, respectively. On OMP electrophoretic profiles, MDRPA showed a reduction of outer membrane porin D (OprD) and PCR demonstrated the presence of a class 1 integron with a cassette encoding aminoglycoside adenyltransferase B (aadB). Meropenem accumulation was slightly higher in bacilli than in the filamentous cells that formed in the presence of antibiotics. Overexpression of the efflux pump MexAB-OprM and a functional MexXY-OprM were detected in all isolates.

One hundred and six nalidixic acid-resistant Enterobacteriaceae isolates from two Brazilian hospitals isolated from June to October 2010 were evaluated to characterize the co-existence of plasmid-mediated quinolone resistant (PMQR) and extended-spectrum β-lactamase (ESBL) determinants. The qnr genetic environment was determined by PCR and sequencing. Conjugation and hybridization experiments determined whether qnr-carrying plasmids were self-transferable. The aac(6′)-Ib-cr and qepA genes were also screened. Thirteen qnr-like genes (12.3 %) were identified, with qnrB1 the most common, followed by qnrS1, qnrB2 and qnrB19. No qnrA, qnrC, qnrD or qepA determinant was detected. All qnr-positive strains possessed chromosomal substitutions in gyrase- and topoisomerase-encoding genes and four harboured a aac(6′)-Ib-cr gene. The co-production of bla CTX-M was observed in ten qnr-positive strains. These results indicate the dissemination of PMQR genes shown in clinical isolates from Brazil, and their co-existence with ESBL genes emphasizes the complexity of plasmid-mediated resistance determinants among Enterobacteriaceae.

This study reports an infectious case involving an NDM-1-producing Citrobacter freundii and further explored the potential threat of the bla NDM-1 gene by analysing the characteristics of the NDM-1-encoding plasmid sequence. A bla NDM-1-positive C. freundii with high resistance to carbapenems was separated from a clinical patient suffering from a urinary tract infection. S1 nuclease-based plasmid analysis followed by Southern blot hybridization, a conjugation experiment and electrotransformation confirmed that the bla NDM-1 gene was located on a plasmid. High-throughput sequencing of the bla NDM-1-postive plasmid (pCFNDM-CN) showed that it was a 54 kb IncX-type plasmid and contained a backbone region and a variable region with two β-lactamase genes (bla NDM-1 and bla SHV-12). The NDM-1 composite transposon in the variable region was surrounded by IS26 and IS5-truncated ISAba125, and shared a high sequence similarity to the bla NDM-1 surrounding structure in Acinetobacter spp. Our research suggested that the NDM-1 composite transposon might play an essential role in mobilization of the bla NDM-1 gene from Acinetobacter spp. to Enterobacteriaceae.

This study analysed the characteristics and genetic similarity of recent Klebsiella pneumoniae carbapenemase (KPC-2)-producing Klebsiella pneumoniae isolates from Korea. Recent laboratory surveillance detected an increase in carbapenemase-producing Enterobacteriaceae in Korea. A total of 6 KPC-2-producing K. pneumoniae were identified from 277 Enterobacteriaceae clinical isolates. All were sequence type (ST) 258 and they had the same pulsotype. They had high MICs for carbapenems and multi-drug resistance. TEM-1, SHV-11 and OXA type β-lactamases were detected in all isolates, whereas CTX-M type β-lactamases and plasmid-mediated AmpC β-lactamase (PABL) were not present. A conjugation experiment failed, but bla KPC-2-harbouring plasmids from the six isolates were used to transform Escherichia coli DH5-α by electroporation. Each of the transformants harboured a bla KPC-2-positive approximately 95 kb plasmid, which was typed in the IncFII incompatibility group and co-harboured TEM-1 and OXA-9 β-lactamases. They shared the same restriction profile. This study confirms the emergence of clonal ST258 KPC-2-producing K. pneumoniae in some regions of Korea.

The objective of this study was to investigate the susceptibility of hospital-associated (HA) and community-associated (CA) Escherichia coli and Klebsiella pneumoniae isolated from patients with intra-abdominal infections (IAIs) in China. From 2002 to 2011, the minimum inhibitory concentrations (MICs) of 12 antibiotics against 3074 E. coli and 1025 K. pneumoniae from 23 centres located in 16 cities were determined by the broth microdilution method. During the 10 year study period, ertapenem, imipenem, amikacin and piperacillin-tazobactam retained high and stable activity against E. coli and K. pneumoniae isolates regardless of whether their source was HA or CA and regardless of their extended-spectrum beta-lactamase (ESBL) production. However, the susceptibility of E. coli to cephalosporins and ampicillin-sulbactam decreased dramatically during the 10 years, especially for the CA isolates. Fluoroquinolones showed low activity against E. coli. During the whole study period, the ESBL rates for E. coli isolates from IAIs increased from 36.1 % in 2002–2003 to 68.1 % in 2010–2011 (P<0.001). Correspondingly, the ESBL rates in HA isolates increased from 52.2 % in 2002–2003 to 70.0 % in 2010–2011 (P = 0.001), and in CA isolates from 19.1 % in 2002–2003 to 61.6 % in 2010–2011 (P<0.001). The ESBL-positive rate in K. pneumoniae remained between 30.1 and 39.3 % of the total isolates with no significant change during the 10 years. In conclusion, carbapenems retained the highest susceptibility rates against HA and CA E. coli and K. pneumoniae. High prevalence of ESBL in HA E. coli and fast-growing resistance in CA E. coli severely limit the empirical use of the third- and fourth-generation cephalosporins in the therapy of IAIs.

In this study, a total of 650 stool samples were tested to show that our method is capable of detecting four Clostridium difficile genes; tcdA, tcdB, encoding toxin A (TcdA) and toxin B (TcdB), and the binary toxin C. difficile transferase genes (cdtA and/or cdtB) encoding CDT toxin. Besides detecting the targeted C. difficile genes, our method can be used to detect the presence of any inhibitory components in the PCR. This assay, combined with a selective culture medium, such as the chromID™ C. difficile, can be applied directly for screening C. difficile-associated disease. The PCR-based assay developed here is rapid (4 h per 21 stool samples) and accurate in diagnosing C. difficile infection, 100 % assay sensitivity and negative predictive value (NPV) were obtained. However, the assay specificity of 99.1 % and positive predictive value (PPV) of 94.9 % were slightly lower than the optimal value of 100 %. The assay protocol outlined here can be used as a rapid screening tool to assist infection control units and in managing infected patients by reducing the number of patients requiring isolation and extended hospitalization. Rapid detection can prevent unnecessary antibiotic therapy and potentially reduce the spread of infection by emerging hypervirulent C. difficile strains.

Vibrio cholerae is a serious public health problem worldwide, but in the UK, V. cholerae infections are rare. Here, we report a case of V. cholerae bacteraemia in an elderly patient. To our knowledge, this is the first non-travel-related V cholerae bacteraemia in the UK.

Salmonella enterica serovar Minnesota is a rarely isolated organism in clinical samples mainly grown from stool cultures. Sepsis due to Salmonella is known in severely immunocompromised patients, but so far urosepsis due to S. enterica serovar Minnesota has not been described. We report a case of a 31-year-old patient suffering from Crohn’s disease treated with infliximab and azathioprine, in whom was implanted a double-J ureteric catheter for urolithiasis. The patient presented with urinary tract infection and severe sepsis. S. enterica serovar Minnesota was grown from urine and blood cultures. After empiric antimicrobial treatment with meropenem and vancomycin, treatment was changed to ceftriaxone. Antimicrobial treatment was continued for a total of 3 weeks without evidence of Salmonella recurrence on follow-up visits. Salmonella spp. rarely cause urinary tract infection and sepsis. However, in immunocompromised patients, non-typhoidal salmonellosis merits a thorough clinical and microbiological evaluation.

Nasal carriage of Staphylococcus aureus is commonly evaluated via culture-based methods. We found that parallel use of two media, Baird-Parker and CHROMagar™ Staph aureus, increased detection of S. aureus from a healthy population by 29 %. We suggest use of both media for optimal identification of S. aureus from healthy cohorts.

Clostridium difficile infection (CDI) is a major cause of morbidity and mortality among hospitalized patients and imposes a considerable financial burden on health service providers in both Europe and the USA. The incidence of CDI has dramatically increased in recent years, partly due to the emergence of a number of hypervirulent strains. The most commonly documented risk factors associated with CDIs are antibiotic usage leading to alterations of the gut microbiota, age >65 years and long-term hospital stay. Since standard therapies for antibiotic-associated diarrhoea and CDI have limited efficacy, there is now an urgent need for alternative therapeutics. In this review, we outline the current state of play with regard to the potential of gut-derived bacteriocins, probiotics and phage to act as antimicrobial agents against CDI in the human gut.

The endospores of Bacillus subtilis can serve as a tool for surface presentation of heterologous proteins. The unique properties of the spore protective layers make them perfect vehicles for orally administered vaccines. In this study, we successfully displayed a fragment of Clostridium difficile FliD protein on the surface of B. subtilis spores using the CotB, CotC, CotG and CotZ spore coat proteins. The presence of the fusion proteins in the spore coat was verified by Western blotting and immunofluorescence microscopy. The amount of recombinant proteins was assessed by a dot-blot technique. C. difficile is one of the most common infectious agents in nosocomial infections and is especially associated with antibiotic therapies. FliD is a flagellar cap protein of C. difficile and is known to be one of the immunogenic surface antigens of this bacterium. Therefore, its use in vaccine formulations gives a good perspective for successful immunization with a FliD-based vaccine. The recombinant spores presented here may be good candidates for C. difficile oral vaccines.