- Volume 64, Issue 10, 2015

Increasing numbers of Campylobacter hyointestinalis have been isolated from humans and animals with gastroenteritis, although the virulence mechanism of this species remains largely unknown. Here, we show that C. hyointestinalis isolated from a patient with diarrhoea in Thailand produced a novel variant of cytolethal distending toxin (CDT). Sequencing of a 13 965 bp genomic region of C. hyointestinalis carrying the genes coding for Ch-CDT revealed three ORFs of 798, 804 and 537 bp, which code for the Ch-CdtA, Ch-CdtB and Ch-CdtC subunits, respectively. The deduced amino acid sequence of Ch-CdtA showed ∼38.9 % homology with the CdtA of Campylobacter coli, but sequences of Ch-CdtB and Ch-CdtC were homologous to CdtB (65.7 %) and CdtC (33.1 %) of Campylobacter upsaliensis, respectively. Filter-sterilized sonic lysate of C. hyointestinalis demonstrated distension and death of HeLa cells by arresting the cell cycle at the G2/M phase and phosphorylation of host histone H2AX, a sensitive marker of DNA double-strand breaks. Rabbit antiserum raised against recombinant Ch-CdtB was not reactive against the recombinant CdtB protein of Campylobacter jejuni. A reconstituted Ch-CDT holotoxin prepared using each of the recombinant subunit proteins demonstrated distension and death of HeLa cells, suggesting that the C. hyointestinalis isolate indeed produced functionally active Ch-CDT. Furthermore, the immunological distinctiveness of the Ch-CDT produced by C. hyointestinalis and the increasing prevalence of the species in patients and animals with gastroenteritis suggest that this species may be an important emerging zoonotic pathogen.

Microflex LT (Bruker Daltonics) and VITEK MS (bioMérieux) are bacterial identification systems that are based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). For VITEK MS, two identification softwares, VITEK MS IVD (IVD) and SARAMIS (SARAMIS), are available. Microflex LT is equipped with MALDI Biotyper RTC software (Biotyper). Although the identification accuracy of each instrument has been compared for various bacteria, no detailed examination has been conducted for the identification accuracy of Clostridium difficile. In this report, we compared the three identification softwares for identification reproducibility in three ATCC C. difficile strains and identification accuracy in 50 clinical C. difficile isolates. The results showed 100, 91.7 and 100 % identification reproducibility accuracy of ATCC strains when examined by IVD, SARAMIS and Biotyper software, respectively. For the identification of the clinical isolates, all three softwares exhibited satisfactory identification accuracy of C. difficile. Among the 50 clinical isolates, seven showed identical toxin genotype corresponding to the exact ribotype. However, MALDI-TOF MS failed to identify them as the identical type. Based on the above results, we concluded that both types of MALDI-TOF MS reproducibly identified C. difficile; however, they are currently not suitable for typing of C. difficile clones.

Acinetobacter baumannii has become one of the major infection threats in intensive care units (ICUs) globally. Since 2008, A. baumannii has been the leading cause of ventilator-associated pneumonia (VAP) in our ICU at an infectious disease hospital in southern Vietnam. The emergence of this pathogen in our setting is consistent with the persistence of a specific clone exhibiting resistance to carbapenems. Antimicrobial combinations may be a strategy to treat infections caused by these carbapenem-resistant A. baumannii. Therefore, we assessed potential antimicrobial combinations against local carbapenem-resistant A. baumannii by measuring in vitro interactions of colistin with four antimicrobials that are locally certified for treating VAP. We first performed antimicrobial susceptibility testing and multilocus variable number tandem repeat analysis (MLVA) genotyping on 74 A. baumannii isolated from quantitative tracheal aspirates from patients with VAP over an 18-month period. These 74 isolates could be subdivided into 21 main clusters by MLVA and >80 % were resistant to carbapenems. We selected 56 representative isolates for in vitro combination synergy testing. Synergy was observed in four (7 %), seven (13 %), 20 (36 %) and 38 (68 %) isolates with combinations of colistin with ceftazidime, ceftriaxone, imipenem and meropenem, respectively. Notably, more carbapenem-resistant A. baumannii isolates (36/43; 84 %) exhibited synergistic activity with a combination of colistin and meropenem than carbapenem-susceptible A. baumannii isolates (2/13; 15 %) (P = 0.023; Fisher's exact test). Our findings suggest that combinations of colistin and meropenem should be considered when treating carbapenem-resistant A. baumannii infections in Vietnam, and we advocate clinical trials investigating combination therapy for VAP.

Of the cephalosporins, cefpodoxime has the most published clinical data for the treatment of urinary tract infections. In 2014, the Clinical and Laboratory Standards Institute (CLSI) guidelines recommended that cefazolin should be used as the surrogate marker for cefpodoxime among urinary tract isolates, replacing cephalothin. This study attempted to determine how well cefazolin serves as the surrogate marker. Additionally, it investigated how cefuroxime compared with cefazolin as a surrogate marker. The MicroScan Walkaway Plus system was used to determine susceptibility for cefazolin and cefuroxime on consecutive urine cultures with a colony count of ≥ 50 000 organisms. Only Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis isolates were included, following CLSI guidelines. Simultaneously, an Etest for cefpodoxime was conducted. The cefpodoxime interpretation was compared with that of the other two agents, and the categorical agreement was calculated, defined as the percentage of identical susceptibility interpretations. Cefazolin (92 %) had a significantly higher categorical agreement than cefuroxime (85 %) among 284 isolates (P = 0.011). The major error rate was 4.4 % for cefazolin and 1.1 % for cefuroxime. The very major error rate was 64 % for cefazolin and 18 % for cefuroxime among the 11 cefpodoxime-resistant isolates. Cefazolin was a better predictor of cefpodoxime susceptibility than the previously recommended agent, cephalothin. However, cefuroxime had better major and very major error rates than cefazolin.

We evaluated temporal fluctuations in macrolide resistance rates, analysing genetic determinants of resistance and clonal evolution in a population of 2744 S. pyogenes isolates collected in the period 2000–2013. The total resistance rate to erythromycin of the isolates was 17.9 %. A maximum of erythromycin resistance emerged in 2000 (38.6 %), followed by a significant decrease to 5.2 % in 2012 (P < 0.0001). Molecular analysis revealed the presence and co-presence of known genetic resistance determinants mefA, mefE, ermTR and ermB, in line with phenotypes. PFGE analysis identified genetically related groups in 2000 and 2007–2008, mainly the MLS and M phenotypes, respectively. The most prevalent emm types among a representative subset of resistant isolates were emm2, emm75 and emm77. All emm2 and 88.2 % of the strains harbouring the emm75 gene were only recorded in M-phenotype strains, whilst all emm77-positive strains had the inducible MLS phenotype. The analysed susceptible isolates showed several emm types partially shared with resistant ones. Our results suggest that changes in bacterial population clonality, rather than horizontal transfer of resistance determinants, plays a major epidemiological role in S. pyogenes. Continuous monitoring of microbiological epidemiology seems to be crucial for correct and effective management of streptococcal infections.

Minocycline (MIN) usually shows good activity against Acinetobacter baumannii strains. The reintroduction to the market of intravenous MIN provides an additional agent to the limited options for the treatment of A. baumannii infections. The activity of MIN as a single agent and in combination with rifampicin (RIF), colistin (COL) or imipenem (IMI) was evaluated by means of killing curves and 24 h-time-kill curves in five A. baumannii isolates which were selected on the basis of different antimicrobial resistance profiles. MIN showed bacteriostatic activity in three isolates (2 ×  or 16 ×  MIC) and bactericidal activity in the other isolates (64 ×  MIC). In isolates harbouring the tetB gene, the associations studied were always indifferent. However, in isolates not harbouring tetB, the use of MIN in combination showed a rapid synergistic effect (at 4 h) in four out of nine combinations (two with RIF and one each with IMI and COL). At 24 h, this effect was observed in six out of nine combinations (two in each association). MIN in combination with RIF, IMI and COL showed bactericidal synergy in most of the isolates which did not harbour the tetB gene, but the combinations were not synergistic in tetB-positive isolates.

Toxoplasmosis is one of the most common parasitic diseases worldwide and it poses a serious challenge regarding prevention, diagnosis and therapy. The commonly used diagnostic methods are mostly based on the detection of specific antibodies in sera. Since they are not always accurate enough and do not allow precise definition of the phase of the Toxoplasma gondii infection, there is an urgent need to find specific molecular markers of acute or chronic infection stages. This study provides a comparative assessment of recombinant ROP5 and ROP18 T. gondii proteins in the serodiagnosis of human toxoplasmosis. We found that both ROP5 and ROP18 proteins allowed the detection of specific IgM and IgG antibodies with a relatively low sensitivity; however, ROP18 IgM ELISA proved to be more sensitive than the SAG1 assay. This study also points to a relatively weak potential of the corresponding native ROP5 and ROP18 kinases in the generation of a strong antibody response in humans.

The epidemiology of nocardiosis is evolving with increasing number of Nocardia spp. causing human infection. In recent years, molecular techniques have been used to identify Nocardia spp. There are limited data available on the spectrum of Nocardia spp. isolated from clinical samples in India. Here, a molecular study was carried on 30 clinical isolates maintained in our National Culture Collection to evaluate the techniques used for identifying the agents. The isolates were identified by sequencing two promising genes: the 16S rRNA gene and hsp65. Both hsp65 and the 16S rRNA gene could reliably identify 90 % of Nocardia isolates, i.e. N. farcinica, N. cyriacigeorgica, N. brasiliensis, N. otitidiscaviarum, N. amamiensis and N. pneumoniae. The mean percentage dissimilarity of sequence identification was higher using the hsp65 gene (4 %, range 0–7.9 %) compared with the 16S rRNA gene (2.3 %, range 0–8.9 %). Two isolates that showed ambiguous results in both the short segment of the 16S rRNA gene and hsp65 sequences could be resolved by sequencing a larger fragment (∼1000 bp) of the 16S rRNA gene. Both of these isolates were identified as N. beijingensis with similarities of 99.8 and 100 % compared with the standard strain. Genotyping of N. cyriacigeorgica strains was performed using hsp65 gene sequences and compared with previously described genotypes. Our N. cyriacigeorgica isolates belonged to genotype 1 (n = 4) and genotype 2 (n = 2). The present study highlights a wide spectrum of Nocardia spp. in India and emphasizes the need for molecular techniques for identification to the species level.

Global spread and evolutionary links of an epidemic Clostridium difficile strain (PCR-ribotype 027) have been noted in recent decades. However, in Japan, no outbreaks caused by type 027 have been reported to date. A total of 120 C. difficile isolates from patients at 15 hospitals during non-outbreak seasons between 2011 and 2013 as well as 18 and 21 isolates collected from two hospitals in 2010 and 2009, respectively, in outbreak periods in Japan, were examined. Among these 120 isolates, Japan-ribotypes smz and ysmz (subtype variant of smz) were the most predominant (39.2 %) followed by Japan-ribotype trf (15.8 %). Types smz/ysmz and trf were also concurrently predominant at two hospitals in the outbreak settings. Out of the five binary toxin-positive isolates observed, only one was PCR-ribotype 027 and another PCR-ribotype 078. Type smz was later found to correspond to PCR-ribotype 018. High rates of resistance against gatifloxacin, moxifloxacin, erythromycin and clindamycin were observed in the PCR-ribotype 018 isolates. Interestingly, all trf isolates were toxin A-negative, toxin B-positive, but they did not correspond to PCR-ribotype 017, thus being assigned a new ribotype (PCR-ribotype 369). In conclusion, PCR-ribotypes 018 (smz) and 369 (trf) were identified as major circulating strains in both outbreak and non-outbreak settings in Japan. Given their epidemiological relevance, molecular investigations are warranted to clarify potential evolutionary links with related strains found elsewhere, such as PCR-ribotypes 018 and 017 from Europe and North America.