- Volume 66, Issue 1, 2017

Propionibacterium acnes is an anaerobic bacterium that causes deep infection in organs and prosthetic joints, in addition to acne vulgaris. Many tetracycline-resistant P. acnes strains have been isolated because oral tetracyclines are frequently used as an acne treatment against P. acnes. In this study, we found a novel tetracycline resistance mechanism in P. acnes. Three doxycycline-resistant (MIC: 16 µg ml−1) strains were isolated from 69 strains in acne patients in Japan between 2010 and 2011. Additionally, six insusceptible strains (MIC: 1–2 µg ml−1) that had reduced susceptibility compared to susceptible strains (MIC: ≤0.5 µg ml−1) were identified. All doxycycline-resistant strains had a G1036C mutation in the 16S rRNA gene in addition to an amino acid substitution in the ribosomal S10 protein encoded by rpsJ. By contrast, insusceptible strains had an amino acid substitution in the S10 protein but no mutation in the 16S rRNA. When the mutant with decreased susceptibility to doxycycline was obtained in vitro, only the mutated S10 protein was found (MIC: 4 µg ml−1), not the mutated 16S rRNA gene. This result shows that the S10 protein amino acid substitution contributes to reduced doxycycline susceptibility in P. acnes and suggests that tetracycline resistance is acquired through a 16S rRNA mutation after the S10 protein amino acid substitution causes reduced susceptibility.

The aim of this study is to determine the frequency of quaternary ammonium compound-resistant genes, including qacA/B, qacC/D, qacE, qacΔE1, qacG, qacJ, and β-lactamase genes, including bla CTX-M, bla TEM, bla SHV, bla OXA-23, bla VIM, bla IMP, bla NDM and bla GIM in 51 isolates of carbapenem-resistant Acinetobacter baumannii collected over a period of 2 years and to determine the MIC of chlorhexidine and quaternary ammonium compound-based disinfectant benzalkonium chloride. The qac and bla genes were detected by the PCR method. The MICs were determined using the broth microdilution method. The MIC of benzalkonium chloride was in the range of 4 to 64 µg l−1 and chlorhexidine in the range of 4 to 64 µg l−1. The qacΔE1 gene was detected in 96.07 % (49/51) isolates and qacE in 31.37 % (16/51), qacG in 23.52 % (12/51) and qacA/B in 13.72 % (7/51). The qacC/D genes and qacJ were not found in any of these strains in this study. The frequency of bla genes was as follows: 84.31 % (43/51) for bla OXA-23, 33.33 % (17/51) for bla TEM, 27.45 % (14/51) for bla VIM, 19.61 % (10/51) for bla SHV, 17.65 % (9/51) for bla NDM, 11.76 % (6/51) for bla GIM and 7.8 % (4/51) for the bla IMP. The bla CTX-M genes were negative in all the strains. From the study, we concluded that reduced susceptibility to the two disinfectants, as well as qac and bla genes, was prevalent among A. baumannii isolates from the hospital environment. This study may offer hospitals important information about the control of nosocomial infections caused by A. baumannii.

Urinary tract infections (UTIs) are clinically important problems that lead to serious morbidity and mortality, and indwelling urinary catheters are a major factor of UTIs. In this study, we applied clustered regularly interspaced short palindromic repeats (CRISPR) genome editing to generate ΔluxS mutant strains from clinical isolates of Escherichia coli SE15, which is one of major pathogens and can cause colonization and biofilm formation in the catheter. A major regulatory pathway of such biofilm formation on medical devices is the quorum sensing mechanism via small molecule autoinducer-2 synthesized by LuxS enzyme. Here, we used the CRISPR-Cas9 system for precise deletion of luxS gene in clinical isolate E. coli SE15. To this end, we constructed a donor DNA for homologous recombination to delete 93 bases in the chromosomal target (luxS) and observed the success rate of luxS deletion to be 22.7 %. We conducted biofilm assay to observe decreased biofilm formation in the E. coil SE15 ΔluxS mutants compared to wild-type E. coil SE15. Quantitative real-time PCR analysis of E. coil SE15 ΔluxS mutants showed that the expression of luxS was below detection level. We also observed that the relative mRNA levels of biofilm-formation-related genes, such as mqsR, pgaBC and csgEF, were significantly decreased in E. coil SE15 ΔluxS mutants compared to wild-type. We conclude that genome editing by CRISPR-Cas9 system is an effective tool to dissect the molecular mechanism of biofilm formation in medically important strains, and the study may serve as a basis for developing novel medical intervention against UTIs caused by biofilm.

Streptococcus agalactiae (group B streptococci, GBS) are important human and animal pathogens, which can be subdivided based on different capsular polysaccharides and surface-anchored alpha-like proteins (Alps), as well as other proteins. Nearly all GBS strains possess an Alp (Alp GBS), although Alp-negative GBS (non-Alp GBS) do occur. In this study, 10 (1.1 %) of 932 clinical human GBS tested lacked an Alp encoding gene. All 10 strains were from patients with bloodstream infection, confirming that non-Alp GBS can be highly virulent. All non-Alp GBS expressed one or more of the surface-anchored proteins R3, Z1 and Z2, while less than 10 % of unselected clinical strains express any of these proteins. In contrast to Alp GBS, all non-Alp strains tested were PCR negative for the upstream sequence of the insertion site of the Alp encoding gene of Alp GBS. Genome sequencing showed that all but one of the 10 clinical non-Alp strains and the non-Alp reference strain CNCTC 10/84 lacked a region surrounding the Alp gene commonly present in Alp GBS strains. These strains instead harboured an 849 bp region not present in the Cα prototype strain A909. We have shown that non-Alp GBS differ from Alp GBS in the region surrounding the insertion site of Alp genes of Alp GBS as well as in their content of other surface proteins and that PCR for the upstream flanking region of the Alp gene may be useful for differentiation between Alp and non-Alp GBS.

Noroviruses, an important cause of diarrhoea in humans, are genetically diverse. The recent norovirus seasons recorded the emergence of new recombinants of the capsid and polymerase genotypes, with a global dominance of GII.Pe_GII.4 Sydney 2012 and GII.P17_GII.17 in Asian countries. However, the number of papers reporting the distribution of both polymerase and capsid genotypes circulating among children is scarce, with none from Vietnam. This study described both the polymerase and capsid genotypes of noroviruses circulating in Vietnamese children using stool specimens obtained under the World Health Organization rotavirus surveillance programme from 2012 to 2015. Of 350 specimens tested, noroviruses were detected in 90 (28 %) of 319 inpatient specimens and in 9 (29 %) of 31 outpatient specimens. The polymerase and capsid genotype combinations of GII.Pe_GII.4 Sydney 2012 and GII.P21_GII.3 were co-dominant (51 and 24 %, respectively), both of which were recombinants, contributing to a high proportion (87 %) of recombinants among circulating noroviruses. GII.4 variants evolved in the same fashion in Vietnam as in other countries, with amino acid substitutions in the putative variant-specific epitopes of the protruding domain. Unlike neighbouring countries where the predominance of GII.P17_GII.17 was reported, only one GII.P17_GII.17 strain was detected from an outpatient in 2015 in Vietnam. In conclusion, a substantial burden due to norovirus gastroenteritis hospitalizations among Vietnamese children was associated with circulating co-dominant GII.Pe_GII.4 Sydney 2012 and GII.P21_GII.3 strains. Continued surveillance is necessary to monitor infection caused by GII.4 variants and that of GII.P17_GII.17 noroviruses in paediatric patients in Vietnam.

Multidrug resistance in the nosocomial pathogen Acinetobacter baumannii limits therapeutic options and impacts on clinical care. Resistance against carbapenems, a group of last-resort antimicrobials for treating multidrug-resistant (MDR) A. baumannii infections, is associated with the expression (and over-expression) of carbapenemases encoded by the bla OXA genes. The aim of this study was to determine the prevalence of antimicrobial-resistant A. baumannii associated with infection in three hospitals in southern Vietnam and to characterize the genetic determinants associated with resistance against carbapenems. We recovered a total of 160 A . baumannii isolates from clinical samples collected in three hospitals in southern Vietnam from 2012 to 2014. Antimicrobial resistance was common; 119/160 (74 %) of isolates were both MDR and extensively drug resistant (XDR). High-level imipenem resistance (>32 µg ml−1) was determined for 109/117 (91.6 %) of the XDR imipenem-nonsusceptible organisms, of which the majority (86.7 %) harboured the bla OXA-51 and bla OXA-23 genes associated with an ISAba1 element. Multiple-locus variable number tandem repeat analysis segregated the 160 A . baumannii into 107 different multiple-locus variable number tandem repeat analysis types, which described five major clusters. The biggest cluster was a clonal complex composed mainly of imipenem-resistant organisms that were isolated from all three of the study hospitals. Our study indicates a very high prevalence of MDR/XDR A. baumannii causing clinically significant infections in hospitals in southern Vietnam. These organisms commonly harboured the bla OXA-23 gene with ISAba1 and were carbapenem resistant; this resistance phenotype may explain their continued selection and ongoing transmission within the Vietnamese healthcare system.

Approximately 20  % of the UK population wear some form of denture prosthesis, resulting in denture stomatitis in half of these individuals. Candida albicans is primarily attributed as the causative agent, due to its biofilm -forming ability. Recently, there has been increasing evidence of C. albicans biofilm heterogeneity and the negative impact it can have clinically; however, this phenomenon has yet to be studied in relation to denture isolates. The aims of this study were to evaluate C. albicans biofilm formation of clinical denture isolates in a denture environment and to assess antimicrobial activity of common denture cleansers against these tenacious communities. C. albicans isolated from dentures of healthy and diseased individuals was quantified using real-time PCR and biofilm biomass assessed using crystal violet. Biofilm development on the denture substratum poly(methyl methacrylate), Molloplast B and Ufi-gel was determined. Biofilm formation was assessed using metabolic and biomass stains, following treatment with denture hygiene products. Although C. albicans was detected in greater quantities in diseased individuals, it was not associated with increased biofilm biomass. Denture substrata were shown to influence biofilm biomass, with poly(methyl methacrylate) providing the most suitable environment for C. albicans to reside. Of all denture hygiene products tested, Milton had the most effective antimicrobial activity, reducing biofilm biomass and viability the greatest. Overall, our results highlight the complex nature of denture- related disease, and disease development cannot always be attributed to a sole cause. It is the distinct combination of various factors that ultimately determines the pathogenic outcome.

Gram-negative ESKAPE pathogens (Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) are responsible for increases in antimicrobial-resistant infections worldwide. We determined in vitro susceptibilities to eight parenteral antimicrobial agents using Clinical and Laboratory Standards Institute broth microdilution methodology for Gram-negative ESKAPE pathogens isolated from hospitalized patients with intra-abdominal infections (IAIs) (n=3052) and urinary tract infections (UTIs) (n=1088) in 11 Asia-Pacific countries/regions from 2013 to 2015. Amikacin (98.3, 96.4 %), imipenem (97.1, 95.5 %) and ertapenem (95.3, 93.2 %) demonstrated the highest rates of susceptibility for isolates of K. pneumoniae from IAI and UTI, respectively, whereas susceptibility to advanced-generation cephalosporins was <84 and <71 %, respectively. K. pneumoniae with an extended-spectrum β-lactamase-positive phenotype were more common in UTI (27.1 %) than IAI (16.2 %). Imipenem and amikacin were the most active agents against extended-spectrum β-lactamase-positive K. pneumoniae from IAI (95.1, 91.8 %) and UTI (94.9, 92.3 %), respectively, whereas <54 % were susceptible to piperacillin–tazobactam. Against Enterobacter spp. and P. aeruginosa, amikacin demonstrated the highest rates of susceptibility for isolates from IAI (99.7, 95.5 %) and UTI (90.9, 91.5 %), respectively. K. pneumoniae, Enterobacter spp. and P. aeruginosa from urine demonstrated lower susceptibility to levofloxacin (74.1, 81.8 and 73.8 %) than from IAI (87.6, 91.8 and 85.4 %). For A. baumannii, rates of susceptibility to all agents tested were <43 %. We conclude that the studied Gram-negative ESKAPE pathogens demonstrated reduced susceptibility to commonly prescribed advanced-generation cephalosporins, piperacillin–tazobactam and levofloxacin, while amikacin and carbapenems were the most active. Ongoing surveillance to monitor evolving resistance trends and the development of novel antimicrobial agents with potent activity against Gram-negative ESKAPE pathogens are mandatory.

The optimal duration of the treatment of ventilator-associated pneumonia (VAP) is still the subject of debate. While 1 week treatment has been reported as possibly sufficient, patients generally receive antibiotic therapy for 10 to 14 days. The purpose of our study was to investigate whether length of treatment in patients with VAP can be reduced with an individualized therapeutic strategy. The study was performed prospectively with patients diagnosed with VAP in our hospital’s intensive care units between 1 January and 31 December 2015. Duration of antibiotic therapy was determined with 5 day clinical evaluation according to previously established criteria. Patients were divided into two groups depending on length of treatment, short (7–10 days) and long treatment (>10 days). Nineteen patients received 7 to 10 day antibiotic therapy, and 30 received >10 day antibiotic therapy. Demographic and clinical characteristics, Glasgow Coma Scale score, CPIS and the PaO2/FiO2 ratio at the time of diagnosis of VAP were statistically similar between the two groups (P>0.05). A second VAP attack occurred post-treatment in three patients receiving short-term treatment and in four receiving long-term treatment (P=0.561). The numbers of antibiotic-free days were 15.6±6.2 in the short-term treatment group and 8.3±7.5 in the long-term group (P<0.0001). One of the patients receiving short-term treatment died within 28 days after treatment, and four of the patients receiving long-term treatment (P=0.348) did so. The most commonly observed micro-organisms in both groups were Acinetobacter baumannii and Pseudomonas aeruginosa. Short-term treatment can be administered in cases with early clinical and laboratory response started on VAP treatment by considering individual characteristics and monitoring fever, CPIS, the PaO2/FiO2 ratio, C-reactive protein and procalcitonin values.

Helicobacter pylori infection is associated with chronic gastritis, peptic ulcers, gastric cancer and mucosa-associated lymphoid tissue lymphoma. The limitations of current therapies for H. pylori infection include poor compliance and antibiotic resistance. Therefore, an effective anti-H. pylori vaccine would be an alternative or complement to antibiotic treatment. Urease B (UreB) is considered an ideal vaccine antigen against H. pylori infection. In this study, cholera toxin B subunit (CTB), a mucosal adjuvant, was used to enhance the immunogenicity of a novel Bacillus subtilis spore vaccine expressing CTB-UreB, along with the B. subtilis spore coat protein CotC as a fusion protein. Oral administration of B. subtilis spores expressing CotC-UreB or CotC-CTB-UreB led to increased levels of UreB-specific IgG in serum and UreB-specific IgA in faeces, as well as elevated levels of IL-10 and IFN-γ in splenocytes. In addition, oral administration of CotC-UreB or CotC-CTB-UreB spores induced significant reductions (80.0 and 90.5 %, respectively) in gastric H. pylori bacterial load (1.11±0.36×105 and 0.53±0.21×105 c.f.u., respectively) compared to that of the CotC control group (5.56±1.64×105 c.f.u., P<0.01). Moreover, CotC-CTB-UreB spores were significantly more effective at reducing the bacterial load than CotC-UreB spores (P<0.05). These results indicate that CotC-CTB-UreB-expressing B. subtilis spores are a potential vaccine candidate for the control of H. pylori infection.

Screening and pre-emptive isolation of at-risk patients are important aspects of the Danish approach to the prevention of meticillin-resistant Staphylococcus aureus (MRSA) infection, but screening with conventional culture can take up to 3 days for results to become available with attendant costs and disadvantages of prolonged isolation. We sought to evaluate the accuracy, time to availability of results and potential economic benefits of two next-generation MRSA screening assays, Xpert MRSA Gen 3 (GX MRSA) and BD MAX MRSA XT, in a setting of a consolidated laboratory serving a number of hospitals with a low prevalence of MRSA and using enrichment culture as a reference method. Four hundred and forty-seven screening samples together with 49 previously positive MRSA samples were evaluated. Xpert MRSA Gen 3 demonstrated sensitivity, specificity, positive predictive value and negative predictive value of 88.2, 97.9, 62.5 and 99.5 %, respectively, and for BD MAX MRSA XT, they were 88.2, 97.4, 57.7 and 99.5 %, respectively. Hands-on time was 8.8 and 21.6 min, respectively, for the Xpert MRSA Gen 3 and BD MAX MRSA XT PCR assays when five samples were handled simultaneously. The mean laboratory turnaround time was 2.9 (1–6) hours for the Xpert MRSA Gen 3 assay, 6.5 (2–46) hours for BD MAX MRSA XT and 49.6 (42–122) hours for enriched culture. Despite laboratory costs being higher for the rapid PCR assays, when the costs of isolation are taken into account, the assays offer the potential for significant cost savings.