- Volume 67, Issue 6, 2018

Purpose. The mechanisms underlying colistin heteroresistance in Acinetobacter baumannii are not fully understood. Here, we investigated the role of efflux in colistin-heteroresistant populations of a multidrug-resistant (MDR) A. baumannii clinical isolate.

Methodology. Three colistin-resistant A. baumannii strain variants isolated from the same clinical sample were studied for the presence of heteroresistance to colistin by drug susceptibility testing, genotyping and drug resistance target mutation analysis. The existence of active efflux was studied by synergism assays with efflux inhibitors, real-time efflux activity measurements and analysis of the mRNA transcriptional levels of selected efflux pump genes in response to colistin.

Results. All of the strain variants belong to the ST218, clonal complex 92, international clonal lineage II. Different colistin susceptibility levels were observed among the three strain variants, indicating that colistin-heteroresistant subpopulations were being selected upon exposure to colistin. No mutations were found in the genes lpxACD and pmrAB, which are associated with colistin resistance. The results showed the existence of synergistic interactions between efflux inhibitors and colistin and ethidium bromide. Real-time efflux assays demonstrated that the three strain variants had increased efflux activity that could be inhibited in the presence of the inhibitors. The efflux pump genes adeB, adeJ, adeG, craA, amvA, abeS and abeM were found to be overexpressed in the strain variants in response to colistin exposure.

Conclusion. This study shows that efflux activity contributes to colistin heteroresistance in an MDR A. baumannii clinical isolate. The use of efflux inhibitors as adjuvants of the therapy can resensitize A. baumannii to colistin and prevent the emergence of drug resistance.

Purpose. To describe the antimicrobial resistance profile of Neisseria meningitidis isolates causing invasive disease in Brazil from 2009 to 2016.

Methodology. Among 3548 N. meningitidis isolates received, 2888 (81.4 %) were analysed for antimicrobial resistance using the broth microdilution technique, as recommended by the Clinical and Laboratory Standards Institute. Isolates were tested for ciprofloxacin, chloramphenicol, ceftriaxone, penicillin G, ampicillin and rifampin.

Results. All the isolates tested were susceptible to ceftriaxone, while 953 (33.0 %), 1307 (45.3 %) and 2 (0.07 %) isolates were penicillin G-, ampicillin- and rifampin-intermediate, respectively. Resistance to rifampin, ciprofloxacin and chloramphenicol was shown by three isolates (0.1 %), two isolates (0.07 %) and one (0.03 %) isolate, respectively. Although no isolates were resistant to penicillin G in the period of 2009–2016, our results show an upward trend in minimum inhibitory concentrations (MICs) for this drug as of 2010 (P<0.001). There was no significant difference between different gender and age groups of patients for reduced susceptibility to penicillin G. There was a higher frequency of isolates with reduced susceptibility to penicillin G in the South and Southeast regions (P<0.001). This reduced susceptibility was also associated with serotype 19 inside serogroup B (P<0.001).

Conclusion. Despite the decrease in susceptibility to penicillin G and ampicillin observed from 2010, the overall resistance of N. meningitidis isolates to the antimicrobials tested remained uncommon and sporadic, confirming their efficacy for chemoprophylaxis or treatment of invasive meningococcal disease (IMD) in Brazil. Continued surveillance of N. meningitidis antimicrobial susceptibility profiles is important in order to monitor variations in resistance either geographically, over time or in association with emergent clones.

Recently, the prevalence of staphylococcal cassette chromosome mec (SCCmec) type IV isolates, which are the major community-acquired methicillin-resistant Staphylococcus aureus (MRSA), have increased in Japanese hospitals. The aim of this study was to elucidate the detailed molecular epidemiological features of the SCCmec type IV clones in Japanese hospitals. When 2589 MRSA isolated from four hospitals in Tokyo, Japan between 2010 and 2014 were analysed, the proportion of SCCmec type IV overtook that of type II, which was the major type of hospital-acquired MRSA in 2014. Multilocus sequence typing showed that CC1 was the most predominant clone in the SCCmec type IV isolates. The clinical departments that the patients belonged to, pulsed-field gel electrophoresis analysis and antimicrobial susceptibility profiles suggested that the origin of the CC1-SCCmec type IV (CC1-IV) clone was a community setting. Our data show that the CC1-IV clone is becoming a predominant MRSA clone in Japanese hospitals.

Purpose. Shiga toxin-producing Escherichia coli (STEC) represents a major issue for public health because of the severity of the associated illnesses, including haemolytic uraemic syndrome (HUS). In 2015, investigation of a case of HUS revealed an outbreak of Shiga toxin-2-producing E. coli O26 : H11 infection in a nursery in Italy. The investigation showed that the infection was transmitted to cases’ contacts via person to person.

Methods. The case finding was performed by testing for STEC stool samples of the HUS case's contacts within the family and the nursery. STEC O26 isolates were characterized by whole genome sequencing. Confirmed cases were repeatedly tested to monitor the duration of STEC shedding.

Results. Eleven STEC O26 cases were identified, including adults and asymptomatic patients. Clinical illness was only observed in children. Strain characterization revealed that a single clone harbouring the stx2a and eae genes and the complete array of STEC-associated virulence genes, belonging to ST(21), was implicated in the outbreak. To reduce bacterial shedding, patients were treated with cefixime following clinical recovery. This antibiotic was well tolerated and did not induce any apparent consequences on patients’ health.

Conclusions. This study confirms that Stx2-producing E. coli O26 represents an emerging public health problem. The occurrence of outbreaks of infection by Stx2-producing E. coli O26 in nurseries is of particular concern, given the high probability of infection progressing in severity and resulting in secondary cases.

Purpose. Candida pathogens are commonly found in women and can cause vulvovaginal candidiasis (VVC), whose infection rate is further increased during pregnancy. We aimed to study the Candida prevalence and strain distribution in pregnant Chinese women with a molecular beacon assay.

Methodology. From March 2016 to February 2017, a total of 993 pregnant women attending routine antenatal visits at the Beijing Obstetrics and Gynecology Hospital were enrolled. For Candida detection and identification, a unique molecular beacon assay was presented and compared with a traditional phenotypic method. Antifungal susceptibility was tested with the following agents: 5-flucytosine, amphotericin B, fluconazole, itraconazole and voriconazole.

Results. The prevalence of Candida was found to be 21.8 % when using the molecular method and 15.0 % when using the phenotypic method. The distribution of the Candida spp. was listed in order of decreasing prevalence: Candida albicans (79.8 %), Candida glabrata (13.5 %), Candida parapsilosis (3.7 %), Candida krusei (2.2 %) and Candida tropicalis (1.1 %). We found that 90.7 % of the Candida detection results were consistent between the molecular and the phenotypic methods. In the cases where the sequencing analyses for the Candida isolates resulted in inconsistent identification, the molecular method showed higher sensitivity than the phenotypic method (96.0 vs 64.6 %). C. albicans, C. glabrata and C. parapsilosis were essentially susceptible to all five antifungal agents tested, whereas C. tropicalis and C. krusei were susceptible to voriconazole and amphotericin B.

Conclusion. By exhibiting good sensitivity and specificity, the molecular assay may offer a fast and accurate Candida screening platform for pregnant women.

Purpose. Acinetobacter baumannii and Pseudomonas aeruginosa are responsible for numerous nosocomial infections. The objective of this study was to determine the development of their susceptibility to ten antibiotics and the antibiotic consumption of patients with suspicion of urinary tract infection (UTI).

Methodology. A retrospective study was conducted on the susceptibility profiles of A. baumannii and P. aeruginosa isolates from 749 urine samples gathered between January 2013 and December 2016, and on the consumption of imipenem, meropenem and piperacillin-tazobactam between 2014 and 2016.

Results. Hospital patients were the source of 82 (91.1 %) of the 90 A. baumannii isolates detected and 555 (84.2 %) of the 659 P. aeruginosa isolates. Globally, the lowest percentage susceptibility values were found for fosfomycin, aztreonam and ciprofloxacin, while colistin continued to be the most active antibiotic in vitro. In 2016, the susceptibility of A. baumannii to carbapenem and piperacillin-tazobactam decreased to very low values, while the susceptibility of P. aeruginosa to carbapenem remained stable but its susceptibility to piperacillin-tazobactam decreased. There was a marked increase in the consumption of piperacillin-tazobactam.

Conclusion. In our setting, it is no longer possible to use carbapenems and piperacillin-tazobactam for empirical treatment of UTI due to A. baumannii or to use piperacillin-tazobactam for empirical treatment of UTI due to P. aeruginosa. Colistin was found to be the most active antibiotic in vitro. There was a marked increase in the consumption of piperacillin-tazobactam.

Purpose. The aim was to assess the performance of both cetylpyridinium chloride (CPC) and OMNIgene•SPUTUM (OMNI) reagents for the maintenance of Mycobacterium tuberculosis viability in sputum prior to recovery by culture.

Methodology. Using 312 sputa, we evaluated the performance of the two reagents using culture on Löwenstein–Jensen medium after sputum storage in CPC or OMNI for up to 28 days. In addition, the viability of M. tuberculosis isolates stored in both reagents was assessed.

Results. The contamination rates for freshly processed samples and those stored in CPC were not statistically different, while the contamination rate for OMNI was significantly lower than that for fresh sputa (P=0.026 for 8 days and P=0.002 for 28 days of storage). The culture positivity for fresh sputa (81.7 %) was similar to that for samples stored in CPC, regardless of the storage time (89.8 % for CPC-8 and 73.0 % for CPC-28). For OMNI-preserved samples, the culture positivity was similar after 8 days of storage (84.2 %), but decreased significantly after 28 days (42.7 %; P<0.0001) compared to fresh sputa, CPC-8, CPC-28 and OMNI-8. There was a significant loss of viability for the H37Rv strain when it was stored in OMNI at room temperature beyond 8 days compared to CPC, but storage at 37 °C decreased recovery from both CPC- and OMNI-stored suspensions.

Conclusion. Culture from sputum stored for 8 days at room temperature in OMNI or CPC gave comparable culture positivity rates to culture from fresh sputum, but after 28 days of storage the performance of OMNI decreased significantly compared to CPC.

Purpose. In this study, we aim to describe and compare the demographical, clinical and laboratory features of leptospirosis and dengue co-infections (LDCI) against single leptospirosis infections in Malaysia.

Methodology. Data of patients admitted to various hospitals in Malaysia from 2011 to 2015 diagnosed with leptospirosis in our laboratory were obtained from their admission records. Co-infection with dengue was determined by collecting dengue serology results. Multivariate analysis and multiple logistic regression were used to differentiate features between single leptospirosis infection and confirmed LDCI.

Results/Key findings. Only 602 (29.11 %) out of 2068 leptospira-positive patients were concurrently tested for dengue during their admission in which 44 (7.31 %) patients had positive non-structural protein 1 (confirmed LDCI) while 140 (23.26 %) were positive for dengue IgM (probable LDCI) with the highest number of cases recorded in high-density suburban districts. Myalgia and arthralgia were the only significant distinguishing clinical feature of LDCI while significant laboratory features were thrombocytopenia and high levels of alanine and aspartate transaminases. Only thrombocytopenia displayed a predictive value for LDCI from analysis of multiple logistic regression. Death occurred in 19 (3.16 %) patients in this dataset studied but only three (0.50 %) were attributed to LDCI.

Conclusion. There is a considerable prevalence of LDCI in this country of which overlapping demographic, clinical and laboratory presentations pose diagnostic and therapeutic challenges. Efforts to raise awareness regarding LDCI, better access to diagnostic services and further prospective studies are warranted.

A plasma metabolomic model obtained by means of untargeted 1H nuclear magnetic resonance, to which taurine, choline, methylamine, total glutathione, trimethylamine N-oxide, lactate, lysine, isoleucine, total fatty acids and unsaturated fatty acids contributed, was validated for the prediction of first episodes of cytomegalovirus (CMV) DNAaemia in a cohort of 79 allogeneic stem haematopoietic stem cell transplant (allo-HSCT) recipients. The predictive success rate was nearly 65 % for patients at both low and high risk of CMV-related complications according to their baseline characteristics. Plasma metabolomics profiling shortly after engraftment (day 21 after transplantation) allowed the anticipation of the occurrence of CMV DNAaemia in 71 % of patients. Plasma metabolomics analyses may be ancillary for identifying allo-HSCT patients at the highest risk of CMV DNAaemia who may benefit from early targeted antiviral prophylaxis.

We evaluated the microscopic and macroscopic characteristics of mycobacteria growth indicator tube (MGIT) cultures for the presumptive identification of the Mycobacterium tuberculosis complex (MTBC) and assessed the reliability of this strategy for correctly directing isolates to drug susceptibility testing (DST) or species identification. A total of 1526 isolates of mycobacteria received at the Instituto Adolfo Lutz were prospectively subjected to presumptive identification by the observation of growth characteristics along with cord formation detection via microscopy. The presumptive identification showed a sensitivity, specificity and accuracy of 98.8, 92.5 and 97.9 %, respectively. Macroscopic analysis of MTBC isolates that would have been erroneously classified as non-tuberculous mycobacteria based solely on microscopic morphology enabled us to direct them rapidly to DST, representing a substantial gain to patients. In conclusion, the growth characteristics of mycobacteria in MGIT, when considered along with cord formation, increased the reliability of the presumptive identification, which has a great impact on the laboratory budget and turnaround times.

Acinetobacter seifertii is a recently described species that belongs to the Acinetobacter calcoaceticus–Acinetobacter baumannii complex. It has been recovered from clinical samples and is sometimes associated with antimicrobial resistance determinants. We present here the case of three A. seifertii clinical isolates which were initially identified as Acinetobacter sp. by phenotypic methods but no identification at the species level was achieved using semi-automated identification methods. The isolates were further analysed by whole genome sequencing and identified as A. seifertii. Due to the fact that A. seifertii has been isolated from serious infections such as respiratory tract and bloodstream infections, we emphasize the importance of correctly identifying isolates of the genus Acinetobacter at the species level to gain a deeper knowledge of their prevalence and clinical impact.

Purpose. The molecular epidemiology and resistance mechanisms of carbapenem-resistant Pseudomonas aeruginosa (CRPA) were determined in hospitals in the countries of the Gulf Cooperation Council (GCC), namely, Saudi Arabia, the United Arab Emirates, Oman, Qatar, Bahrain and Kuwait.

Methodology. Isolates were screened for common carbapenem-resistance genes by PCR. Relatedness between isolates was assessed using previously described genotyping methods: an informative-single nucleotide polymorphism MassARRAY iPLEX assay (iPLEX20SNP) and the enterobacterial repetitive intergenic consensus (ERIC)-PCR assay, with selected isolates being subjected to multilocus sequence typing (MLST). Ninety-five non-repetitive isolates that were found to be resistant to carbapenems were subjected to further investigation.

Results/Key findings. The most prevalent carbapenemase-encoding gene, bla VIM-type, was found in 37/95 (39 %) isolates, while only 1 isolate (from UAE) was found to have bla IMP-type. None of the CRPA were found to have bla NDM-type or bla KPC-type. We found a total of 14 sequence type (ST) clusters, with 4 of these clusters being observed in more than 1 country. Several clusters belonged to the previously recognized internationally disseminated high-risk clones ST357, ST235, ST111, ST233 and ST654. We also found the less predominant ST316, ST308 and ST823 clones, and novel MLST types (ST2010, ST2011, ST2012 and ST2013), in our collection.

Conclusion. Overall our data show that ‘high-risk’ CRPA clones are now detected in the region and highlight the need for strategies to limit further spread of such organisms, including enhanced surveillance, infection control precautions and further promotion of antibiotic stewardship programmes.

Purpose. We recently showed that enteroviruses (EVs) andenterovirus species C (EV-C) in particular were abundant in faecal samples from children who had been diagnosed with acute flaccid paralysis (AFP) in Nigeria but declared to be EV-free by the RD-L20B cell culture-based algorithm. In this study, we investigated whether this observed preponderance of EVs (and EV-Cs) in such samples varies by geographical region.

Methodology. One hundred and eight samples (i.e. 54 paired stool suspensions from 54 AFP cases) that had previously been confirmed to be negative for EVs by the WHO-recommended RD-L20B cell culture-based algorithm were analysed. The 108 samples were made into 54 pools (27 each from North-West and South-South Nigeria). All were subjected to RNA extraction, cDNA synthesis and the WHO-recommended semi-nested PCR assay and its modifications. All of the amplicons were sequenced, and the enteroviruses identified, using the enterovirus genotyping tool and phylogenetic analysis.

Results. EVs were detected in 16 (29.63 %) of the 54 samples that were screened and successfully identified in 14 (25.93 %). Of these, 10 were from North-West and 4 were from South-South Nigeria. One (7.14 %), 2 (14.29 %) and 11 (78.57 %) of the strains detected were EV-A, EV-B and EV-C, respectively. The 10 strains from North-West Nigeria included 7 EV types, namely CV-A10, E29, CV-A13, CV-A17, CV-A19, CV-A24 and EV-C99. The four EV types recovered from South-South Nigeria were E31, CV-A1, EV-C99 and EV-C116.

Conclusion. The results of this study showed that the presence of EVs and consequently EV-Cs in AFP samples declared to be EV-free by the RD-L20B cell culture-based algorithm varies by geographical region in Nigeria.

We compared the genomes of 60 isolates of enteroinvasive Escherichia coli (EIEC) in order to better understand the step-wise evolutionary process from non-pathogenic to host-adapted pathogenic E. coli. All isolates belonged to either sequence type (ST) 6, ST99 or ST270. Each ST was located on different branches of the E. coli phylogeny and had invasion plasmids (pINVs) belonging to FII-21 (ST99, ST270), FII-27 (ST270) or FII-28 (ST6, ST270) incompatibility groups. A higher number of insertion sequence (IS) elements were identified in ST6 and ST270 than in ST99, and appeared to be driving the loss of functional genes. Comparison of the pINV from each ST revealed different degrees of gene loss, with pINV from ST270 being most similar to that found in Shigella species. We captured three EIEC STs at different stages of patho-adaptation, with ST270 being the most ‘shigella-like’ and the most divergent from non-pathogenic E. coli, and ST99 being the least divergent.