- Volume 68, Issue 2, 2019

The modified carbapenem inactivation method (mCIM) is a simple phenotypic screening method for detecting carbapenemase production by Enterobacteriaceae and Pseudomonas aeruginosa . We recently developed another modified carbapenem inactivation method (CIMTris), in which carbapenemase is extracted from bacteria with Tris-HCl buffer, to detect carbapenemase production by Acinetobacter and Pseudomonas species. This study describes an improved carbapenem inactivation method, CIMTrisII, for detecting carbapenemase production by Gram-negative pathogens, including Enterobacteriaceae , Acinetobacter and Pseudomonas species. CIMTrisII was different from CIMTris in the concentration of Meropenem disks (5-µg MEM disks vs. 10-µg MEM disks), the inoculum volume of the bacteria (a 5-µl loopful vs. a 10 µl loopful) and the incubation time (1 vs. 2 h). CIMTrisII showed an overall sensitivity of 99.3 % and an overall specificity of 95.0 % for tested isolates. In comparison, CIMTris showed a sensitivity of 96.1 % and a specificity of 96.3 %, and mCIM showed a sensitivity of 67.1 % and a specificity of 100 %. CIMTrisII is thus deemed useful for detecting carbapenemase production by Gram-negative pathogens.

A number of national and international organizations are advocating more intensive screening for Chlamydia trachomatis and Neisseria gonorrhoeae in high-prevalence populations as a way to reduce the prevalence of these infections. In this article, we review the available evidence and conclude that there is a paucity of evidence to support this approach. We further hypothesize that increasing screening intensity in high-prevalence populations will result in a considerable risk for the emergence of antimicrobial resistance in Neisseria gonorrhoeae and other pathobionts.

Purpose. This study was aimed at investigating the occurrence and genetic mechanisms of resistance to ciprofloxacin, tetracycline and erythromycin in clinical isolates of Campylobacter jejuni recovered from human cases of acute gastroenteritis in Turkey.

Methodology. MIC values of each antibiotic were determined with the epsilometer test (E-test). Resistance genes/mutations were first screened by PCR and analysed by subsequent DNA sequencing.

Results. From a total of 152 C. jejuni isolates tested, 113 (74.3%), 38 (25%) and 9 (5.9%) were found to be resistant to ciprofloxacin, tetracycline and erythromycin, respectively. Sequence analysis of ciprofloxacin-resistant isolates showed that all resistant strains (n=113) carried Thr-86-Ile substition in the gyrA gene, which is the most frequently observed mutation in fluoroquinolone-resistant Campylobacter . All of the tetracycline-resistant isolates (n=38) carried the tetO gene. All of the erythromycin-resistant isolates (n=9) harboured the point mutation A2075G in the 23S rRNA gene, which is the most common mutation conferring macrolide resistance in C. jejuni .

Conclusion. The phenotypic susceptibility testing results were found to agree well with those obtained by genetic detection methods for the C. jejuni isolates tested. The findings of this study showed a very high level of resistance to ciprofloxacin and to a lesser extent to tetracycline while resistance to erythromycin remained at a low level. Thus, erythromycin may be considered as the first choice for treatment of Campylobacter infections in this geographical region when indicated.

Purpose. Staphylococcus aureus isolates, collected from various clinical samples, were analysed to evaluate the contribution of the genetic background of both erythromycin-resistant (ERSA) and -susceptible (ESSA) S. aureus strains to biofilm formation.

Methods. A total of 66 ESSA and 43 ERSA clinical isolates were studied for adhesiveness and biofilm formation under different atmospheres. All isolates were evaluated for phenotypic and genotypic macrolide resistance, and for clonal relatedness by pulsed-field gel electrophoresis (PFGE), and by spa typing on representative isolates.

Results. A high genetic heterogeneity was encountered, although 10 major PFGE types accounted for 86 % with a few small spatially and temporally related clusters. Overall, biofilm formation under anoxia was significantly lower than under oxic and micro-aerophilic atmospheres. Biofilm formation by ESSA was significantly higher compared to ERSA under oxic and micro-aerophilic conditions. Adhesiveness to plastic was significantly higher among respiratory tract infection isolates under micro-aerophilic conditions, while surgical site infection isolates formed significantly higher biomass of biofilm under oxic and micro-aerophilic atmospheres compared to anoxia. Pulsotype 2 and 4 strains formed significantly higher biofilm biomass than pulsotype 1, with strains belonging to CC8 forming significantly more compared to those belonging to CC5, under both oxic and micro-aerophilic atmospheres.

Conclusions. S. aureus biofilm formation appears to be more efficient in ESSA than ERSA, associated with specific S. aureus lineages, mainly CC8 and CC15, and affected by atmosphere. Further studies investigating the relationship between antibiotic resistance and biofilm formation could prove useful in the development of new strategies for the management of S. aureus infections.

Purpose. With an increase in the numbers of bacterial isolates resistant to first-line antibiotics, there has been a revival in the use of older drugs including fosfomycin with novel mechanisms of action. We aimed to investigate the prevalence and genotypic nature of fosfomycin resistance in Escherichia coli from urinary tract infections (UTIs) using the various methods available in the clinical microbiology laboratory.

Methodology. In total, 1000 culture-positive urine samples were assessed for the presence of E. coli and fosfomycin susceptibility was determined using the MAST Uri system, microbroth dilution, agar dilution and E-test strips.

Results/Key findings. Initial investigation using breakpoint susceptibility testing on the MAST Uri system identified 62 of 657 (9.5 %) E. coli isolates as fosfomycin-resistant (MIC≥32 µg ml−1). However, on further testing, a lower rate of eight of the 62 (1.3 %) were robustly confirmed to be resistant using microbroth dilution, agar dilution and E-test strips. These true resistant isolates belonged to diverse E. coli multi-locus sequence types and each had a unique set of chromosomal alterations in genes associated with fosfomycin resistance. Fosfomycin-resistant isolates were not multiply drug resistant and did not carry plasmidic fosfomycin resistance genes. Therefore, the use of fosfomycin may be unlikely to drive selection of a particular clone or movement of transferrable resistance genes.

Conclusion. Fosfomycin remains a viable option for the treatment of E. coli in uncomplicated UTIs; different susceptibility testing platforms can give very different results regarding the prevalence of fosfomycin resistance, with false positives being a potential problem that may unnecessarily limit the use of this agent.

Purpose. In infectious disease therapy, administration of adequate antimicrobial agents is essential for preventing the emergence and spread of resistant bacteria. However, conventional antimicrobial susceptibility testing (AST), based on bacterial growth, is time consuming; therefore, a rapid, simple assay is needed for the timely selection of appropriate antibiotics in clinical laboratories. Here, we established a simple, cost-effective, time-saving and highly sensitive AST assay based on loop-mediated isothermal amplification (LAMP).

Methodology. The targeted bacteria were cultivated for a short period with or without antibiotic before the LAMP reaction. The time to detect a positive reaction with LAMP was used to generate a threshold time (Tt) value, and subtraction of the Tt value for an antibiotic-free sample from the Tt value in an antibiotic-exposed sample generated the ΔTt value, which was used as a marker of antimicrobial susceptibility. The ΔTt value generated using the LAMP-based assay simply and quickly detected antimicrobial resistance in clinical Escherichia coli isolates.

Results. Detection of susceptibility to levofloxacin using the ΔTt value perfectly matched with the results of the conventional assay. In addition, the sensitivity and specificity for the detection of ampicillin, trimethoprim-sulfamethoxazole and fosfomycin resistance were 100 %, 93.8 %, 100 % and 80.0 %, 93.3 %, 97.6 %, respectively.

Conclusion. These results showed that this LAMP-based AST has high sensitivity and specificity for detecting resistant strains and a significant time advantage compared with the conventional method.

Purpose. Escherichia coli is a leading cause of bloodstream infection (BSI) in hospitals and communities.

Methodology. We conducted a retrospective study in 2015 to evaluate the clinical features and microbiological characteristics of E. coli BSI acquired in the hospital and community.

Results. A total of 100 patients with E. coli BSI were enrolled, among whom 60 % had hospital-onset (HO) BSI while 40 % had community-onset (CO) BSI. Patients with HO BSI had higher percentages of haematological disorders, immunosuppression conditions, underwent surgery within 2 weeks and had a higher 30-day mortality. The prevalences of multidrug-resistant and extended-spectrum β-lactamase-producing strains were 81 and 60 %, respectively. Resistance percentages to ampicillin, ampicillin-sulbactam, cefazolin, ceftriaxone, ciprofloxacin and levofloxacin were greater than 50 %. Of the 43 different sequence types (STs) identified, ST131 (15.3 %) was the most common. The serum agglutination rate was 52 % in which 13 O and 11 H serogroups were observed. Among the 36 detected virulence factor (VF) genes, IutA (66 %) and traT (61 %) were the most predominant. papA, papC and papEF were different between the CO and HO BSI groups. VF scores were high (mean >7) in the frequently detected ST95, ST1193 and ST131.

Conclusion. This study revealed that the clinical features of HO and CO E. coli BSI were different. STs and serotypes showed a great diversity in this region while VF genes of the isolates varied between clones.

Purpose. Members of the ST127 uropathogenic E. coli (UPEC) clone have a high virulence potential and are also highly virulent in insect infection models. However, strains of this lineage are reported in relatively low numbers in many studies. ST127 strains are also usually widely susceptible to antibiotics and, consequently, their true prevalence may be under-recognized as they will be eradicated during empirical therapy. A genuine concern is the possibility that members of this highly virulent lineage will acquire resistance, leading to a more serious threat. The aim of this study was to design and validate a PCR assay specific to ST127.

Methodology. Genomic sequences obtained from various UPEC isolates from the leading clones were used in comparative genomic analyses to allow identification of highly discriminatory sequences specific to E. coli ST127. The fliC (flagellin) and a homologue of the upaG (autotransporter adhesin) gene were identified as meeting our criteria and were used to develop a multiplex PCR assay. A total of 143 UPEC isolates representing 99 different MLST clones from three locations (North West and South West England and Riyadh, Saudi Arabia) were used to validate the PCR assay.

Results. The multiplex PCR readily identified all 29 E. coli ST127 isolates but, equally importantly, produced no false positives with representatives of any of the other 98 STs tested.

Conclusion. We report the design and validation of a specific multiplex PCR for the rapid and reliable identification of ST127, which can be used for enhanced surveillance for this high-risk clone.

The Roche Cobas 6800 CT/NG assay was compared to the Abbott m2000 Real Time CT/NG assay for detecting Chlamydia trachomatis and Neisseria gonorrhoeae in 714 specimens referred to the bacteriology laboratory at Geneva University Hospitals, between November 2017 and March 2018, and in nine external quality controls for molecular diagnostics (seven from QCMD Glasgow and two from UK NEQAS). For C. trachomatis , the sensitivity of C6800 compared to m2000 was 100 % (95 % confidence interval [CI], 97.5 to 100 %), the specificity was 99.1 % (95 % CI, 98.0 to 99.7 %). For N. gonorrhoeae , the sensitivity of the C6800 compared to m2000 was 100 % (95 % CI, 90.5 to 100 %), whereas the specificity was 99.7 % (95 % CI, 98.9 to 99.9 %). The C6800 CT/NG assay appears to perform with great accuracy the detection of C. trachomatis and N. gonorrhoeae .

Between September 2013 and March 2016, 26 (1.95 %) of 1333 Staphylococcus aureus clinical isolates from a Chinese hospital were found to be resistant to mupirocin, including 18 (1.35 %) with high-level mupirocin resistance and 8 (0.6 %) with low-level mupirocin resistance. Among the 18 isolates with high-level mupirocin resistance, 17 were associated with plasmid-mediated mupA. Meanwhile, the 8 isolates with low-level mupirocin resistance were shown to have a V588F mutation in ileS. A total of 14 sequence types (STs) and 18 spa types were identified. All four isolates with t062 belonged to ST965. Three ST5-MRSA-SCCmec II were linked to t311, which was not previously reported. Furthermore, ST764-MRSA-SCCmec II-t002, exclusively found in Japan before, was identified in this study. In conclusion, we observed relatively low prevalence of mupirocin resistance among S. aureus with considerable heterogeneity in East China. Newly emerging MRSA clones with high-level mupirocin resistance should be of concern.

Purpose. Mycoplasma hominis is considered among the causes of urogenital infections and shows increasing resistance to fluoroquinolones. However, data regarding the fluoroquinolone resistance mechanism of M. hominis in Southwest China are limited. This study aimed to investigate gene mutations of quinolone resistance-determining regions (QRDRs) of M. hominis isolated from clinical urogenital samples in a Chinese hospital.

Methodology. Strains of M. hominis were identified by 16S rRNA gene sequencing. The minimal inhibitory concentrations (MICs) of fluoroquinolones were determined by the broth microdilution method, following CLSI guidelines. PCR was used to amplify the QRDRs of the genes gyrA, gyrB, parC and parE. Positive products were sequenced, and gene mutations and amino acid substitutions were analysed by DNAMAN software and BLAST.

Results. The resistance rates of M. hominis to ciprofloxacin (CIP), levofloxacin (LVX), moxifloxacin (MXF) and gatifloxacin (GAT) were 90.5, 85.7, 73.8 and 71.4 %, respectively. A total of 57 isolates of M. hominis were screened, among which 52 strains demonstrated different resistant phenotypes to fluoroquinolones, 41 harboured amino acid substitutions of GyrA S153L, 51 harboured ParC S91I and 22 harboured ParC K144R. ParE A463S and ParC A154T were recorded for the first time and no amino acid change was detected in GyrB.

Conclusion. The resistance of M. hominis to fluoroquinolones in Southwest China is mainly related to mutations in QRDRs of either gyrA or parC. High-level resistance is associated with mutations in both DNA gyrase and topoisomerase IV.

Tuberculosis (TB) remains a major threat to human health worldwide. The increasing incidence of non-tuberculous mycobacterial infections and particularly those produced by Mycobacterium avium has emphasized the need to develop new drugs. Additionally, high levels of natural drug resistance in non-tuberculous mycobacteria (NTM) and the emergence of multidrug-resistant (MDR) TB is of great concern. Antimicrobial peptides (AMPs) are antibiotics with broad-spectrum antimicrobial activity. The objective was to assess the activity of AMPs against Mycobacterium tuberculosis and M. avium clinical isolates. MICs were determined using microtitre plates and the resazurin assay. Mastoparan and melittin showed the greatest activity against M. tuberculosis , while indolicidin had the lowest MIC against M. avium . In conclusion, AMPs could be alternatives for the treatment of mycobacterial infections. Further investigation of AMPs' activity in combination and associated with conventional antibiotics and their loading into drug-delivery systems could lead to their use in clinical practice.

Purpose. Mycobacteria are common causative agents of bacterial infections in many species of freshwater and marine fish. Identification of mycobacteria to the species level based on phenotypic tests is inappropriate and time consuming. Molecular methods such as partial or entire gene sequence determination in mycobacteria have been employed to resolve these problems. The objective of this study was to assess the use of sequence analysis of the mycobacterial 16S–23S internal transcribed spacer (ITS) region for the identification of different aquatic mycobacteria species.

Methodology. Using published primers, the ITS sequences of 64 field and reference strains were determined.

Results/Key findings. The identity of all isolates previously identified as Mycobacterium marinum by RFLP was confirmed as M. marinum by sequence analysis. With the exception of five rapidly growing mycobacteria isolates, all other mycobacteria were easily identified by sequencing of the ITS region. Using this spacer region, it was possible to differentiate between slowly growing and rapidly growing mycobacteria, even before sequence analysis, by the size of the PCR product, although species identification could not be made by size alone.

Conclusion. Overall, direct sequencing of this genetic element following PCR has been shown to be useful in the identification of aquatic mycobacteria species. With regard to the variability of the ITS region for different mycobacteria isolates, this may be a useful tool in epidemiological studies.

Purpose. Sporothrix globosa is the most important agent of sporotrichosis in China. The aim of this study is to investigate the population parameters of S. globosa.

Methodology. In the present study, we developed a set of microsatellite markers that have a cumulative discriminatory power of 1.000. Using these microsatellite loci, 120 strains of S. globosa that had clear sampling information were analysed.

Results. Population structure analyses revealed that S. globosa can be separated into three clusters. Analysis of molecular variance (AMOVA) results indicated that genetic variation was more significant among these three clusters than between the two clinical types analysed. In addition, cluster II might have the widest range of distribution and contain higher genetic diversity than the other clusters.

Conclusions. Our work is the first to develop a suite of highly discriminatory microsatellite markers and reveal the population parameters of S. globosa, and our results suggest that different lineages can coexist in two different clinical types. In addition, it was hypothesised that lineages with higher genetic diversity might have a wider distribution range.