- Volume 67, Issue 5, 2018

Purpose. Ceftriaxone is the drug of choice for typhoid fever and the emergence of resistant Salmonella Typhi raises major concerns for treatment. There are an increasing number of sporadic reports of ceftriaxone-resistant S. Typhi and limiting the risk of treatment failure in the patient and outbreaks in the community must be prioritized. This study describes the use of whole genome sequencing to guide outbreak identification and case management.

Methodology. An isolate of ceftriaxone-resistant S. Typhi from the blood of a child taken in 2000 at the Popular Diagnostic Center, Dhaka, Bangladesh was subjected to whole genome sequencing, using an Illumina NextSeq 500 and analysis using Geneious software.

Results/Key findings. Comparison with other ceftriaxone-resistant S. Typhi revealed an isolate from the Democratic Republic of the Congo in 2015 as the closest relative but no evidence of an outbreak. A plasmid belonging to incompatibility group I1 (IncI1-ST31) which included bla CTX-M-15 (ceftriaxone resistance) associated with ISEcp-1 was identified. High similarity (90 %) was seen with pS115, an IncI1 plasmid from S. Enteritidis, and with pESBL-EA11, an incI1 plasmid from E. coli (99 %) showing that S. Typhi has access to ceftriaxone resistance through the acquisition of common plasmids.

Conclusions. The transmission of ceftriaxone resistance from E. coli to S. Typhi is of concern because of clinical resistance to ceftriaxone, the main stay of typhoid treatment. Whole genome sequencing, albeit several years after the isolation, demonstrated the success of containment but clinical trials with alternative agents are urgently required.

The aim of this study was to characterize the presence of carbapenemase-encoding genes in distinct species of Acinetobacter spp. isolated from Brazilian hospitals. Five carbapenem-resistant Acinetobacter spp. isolates (two Acinetobacter pittii, two Acinetobacter bereziniae and one Acinetobacter junii) recovered from two distinct hospitals between 2000 and 2016 were included in this study. All of the isolates harboured bla IMP-1, which was inserted into In86, a class 1 integron. Pulsed field gel eletrophoresis analysis showed that both A. pittii were identical, while the two A. berezinae isolates were considered to be clonally related. In this study, we demonstrated that mobile elements carrying carbapenemase-encoding genes such as In86 may persist for a long period, allowing their mobilization from A. baumannii to other Acinetobacter spp. that are usually susceptible to multiple antimicrobials.

Purpose. Adherence capacity is one of the principal virulence factors of Streptococcus mutans, and adhesion virulence factors are controlled by small RNAs (sRNAs) at the post-transcriptional level in various bacteria. Here, we aimed to identify and decipher putative adhesion-related sRNAs in clinical strains of S. mutans.

Methodology. RNA deep-sequencing was performed to identify potential sRNAs under different adhesion conditions. The expression of sRNAs was analysed by quantitative real-time PCR (qRT-PCR), and bioinformatic methods were used to predict the functional characteristics of sRNAs.

Results. A total of 736 differentially expressed candidate sRNAs were predicted, and these included 352 sRNAs located on the antisense to mRNA (AM) and 384 sRNAs in intergenic regions (IGRs). The top 7 differentially expressed sRNAs were successfully validated by qRT-PCR in UA159, and 2 of these were further confirmed in 100 clinical isolates. Moreover, the sequences of two sRNAs were conserved in other Streptococcus species, indicating a conserved role in such closely related species. A good correlation between the expression of sRNAs and the adhesion of 100 clinical strains was observed, which, combined with GO and KEGG, provides a perspective for the comprehension of sRNA function annotation.

Conclusion. This study revealed a multitude of novel putative adhesion-related sRNAs in S. mutans and contributed to a better understanding of information concerning the transcriptional regulation of adhesion in S. mutans.

Purpose. Group B streptococcus (GBS) is an important cause of neonatal sepsis worldwide. Data on the prevalence of maternal GBS colonization, risk factors for carriage, antibiotic susceptibility and circulating serotypes are necessary to tailor adequate locally relevant public health policies.

Methodology. A prospective study including pregnant women and their newborns was conducted between March and July 2013 in Morocco. We collected clinical data and vagino-rectal and urine samples from the recruited pregnant women, together with the clinical characteristics of, and body surface samples from, their newborns. Additionally, the first three newborns admitted every day with suspected invasive infection were recruited for a thorough screening for neonatal sepsis. Serotypes were characterized by molecular testing.

Results. A total of 350 pregnant women and 139 of their newborns were recruited. The prevalence of pregnant women colonized by GBS was 24 %. In 5/160 additional sick newborns recruited with suspected sepsis, the blood cultures were positive for GBS. Gestational hypertension and vaginal pruritus were significantly associated with a vagino-rectal GBS colonization in univariate analyses. All of the strains were susceptible to penicillin, while 7 % were resistant to clindamycin and 12 % were resistant to erythromycin. The most common GBS serotypes detected included V, II and III.

Conclusion. In Morocco, maternal GBS colonization is high. Penicillin can continue to be the cornerstone of intrapartum antibiotic prophylaxis. A pentavalent GBS vaccine (Ia, Ib, II, III and V) would have been effective against the majority of the colonizing cases in this setting, but a trivalent one (Ia, Ib and III) would only prevent 28 % of the cases.

Purpose. The aim of this study was to evaluate the in vitro activity of double-carbapenem combinations against OXA-48-producing Klebsiella pneumoniae clinical isolates.

Methodology. Double combinations of ertapenem, meropenem and imipenem were evaluated for synergy and bactericidal activity using the time–kill methodology. All antibiotics were tested at 10 mg l−1 and at a sub-inhibitory concentration of 0.5× minimum inhibitory concentration (MIC) for isolates with a carbapenem MIC≤8 mg l−1. Synergy was defined as a ≥2log10 colony-forming units (c.f.u.) ml−1 decrease of viable colonies at 24 h compared to the most active carbapenem alone.

Results. Ten distinct K. pneumoniae clinical isolates were tested. All carried bla OXA-48 and bla CTX-M-15, and exhibited an MIC range of 64–128, 4–32 and 1–32 mg l−1 for ertapenem, meropenem and imipenem, respectively. Out of 48 isolate-combinations, synergy was observed in 9 (18.8 %) and cidal activity was observed in 13 (27.1 %). In vitro synergistic activity was noted for 5 out of 29 (17.2 %) ertapenem-, 6 out of 29 (20.7 %) meropenem- and 7 out of 38 (18.4 %) imipenem-containing combinations. No combination exhibited antagonism. Bactericidal activity was observed in 7 (24.1 %) ertapenem-, 8 (27.6 %) meropenem- and 11 (28.9 %) imipenem-containing combinations. Among the sub-inhibitory concentration combinations, three (15 %) ertapenem-, four (20 %) meropenem- and three (15 %) imipenem-containing ones showed synergistic interaction.

Conclusion. Dual combinations of carbapenems, including those containing sub-inhibitory concentrations of antibiotics, were synergistic against multidrug-resistant (MDR) and extensively drug-resistant (XDR) K. pneumoniae isolates harbouring bla OXA-48.

Purpose. Cytological and histological cervical screening methods for human papillomavirus may be subjective. Current guidelines recommend the use of direct human papillomavirus screening by molecular methods in conjunction with cytology for the detection of high-risk human papillomavirus types with carcinogenic potential. In this study, we compared the performance of the molecular Cepheid Xpert HPV test to the FDA-approved HC2 High-Risk HPV DNA Test on samples from patients presenting for cervical screening, regardless of the cytology results, in which cervical cell samples were originally collected for Papanicolaou (Pap) smear specimens in Becton Dickinson (BD) SurePath preservative fluid.

Methodology. Cervical cells were obtained for Pap smear specimens from 343 women attending Qatif Central Hospital in Saudi Arabia for cervical cancer screening using a Cytobrush Plus GT and immersed in BD SurePath preservative fluid in BD SurePath collection vials. The study was carried out between December 2015 and July 2016.

Results. The Xpert HPV test was positive in 27 (7.9 %) of the samples. The HC2 High-Risk HPV DNA Test was positive in 32 (9.3 %) of the samples. The most common HPV types according to the Xpert HPV test were HPV other types, either alone (n=15) or in combination with HPV16 (n=3). The overall concordance rate between the tests was 98.5 %. The positive concordance was 84.4 %.

Conclusion. The Xpert HPV test is convenient to use on cervical cell samples collected for Pap smear specimens in BD SurePath preservative fluid within an hour and is a viable alternative to the HC2 High-Risk HPV DNA Test for HPV testing.

Brucellosis is a challenging zoonosis to diagnose and treat. The recent outbreaks of Brucella abortus RB51 in several states in the United States, including New Jersey and Texas, due to raw milk consumption has raised the concern of drug-resistant Brucella spp. This commentary highlights the importance of being on the lookout for this emerging infection.

Accurate identification of Mycobacterium tuberculosis complex (MTBC) isolates is essential for tuberculosis (TB) control, especially in a high-burden country such as Brazil. Conventional identification methods are laborious and time-consuming, while rapid molecular methods are expensive and require skilled personnel and appropriate physical laboratory infrastructure. Immunochromatographic assays (ICAs) have been shown to provide a rapid and reliable TB diagnosis at a low cost. The use of the SD Bioline TB Ag MPT64 ICA (MPT64 assay) for rapid identification of MTBC clinical isolates in the routine diagnosis of a large-volume reference TB laboratory was evaluated. We analysed 375 isolates on solid and liquid media concurrently with conventional phenotypic methods, the PRA-hsp65 molecular technique and the MPT64 assay. The sensitivity, specificity and accuracy of the ICA were 97.7, 100 and 98.1 %, respectively. The MPT64 assay yielded rapid and accurate results, enabling the treatment to be initiated early and also impacting on TB control.

Purpose. β-lactamase-negative ampicillin-resistant (BLNAR) Haemophilus influenzae is frequently isolated from respiratory samples and is particularly problematic in Japan. The aim of this study was to characterize circulating isolates of H. influenzae genotypically by BLNAR-PCR and multilocus sequence typing (MLST), and to determine any associations between them.

Methods. H. influenzae isolates (n=191) were collected from paediatric patients (1 month to 12 years old) between 2000 and 2011 for three types of infections: pneumonia (n=61), acute otitis media (AOM) (n=68) and meningitis (n=62). All were characterized for capsular type by agglutination tests, and for β-lactam resistance by real-time PCR. The sequence types (STs) determined by MLST were analysed using eburst v3.

Results. Eighty-eight out of 191 (46.1 %) H. influenzae isolates were BLNAR by PCR; 37 of 61 (60.7 %) from pneumonia; 33 of 68 (48.5 %) from AOM and 18 of 62 (29.0 %) from meningitis cases. MLST identified 40 and 44 STs among isolates from pneumonia and AOM, respectively. BLNAR were found in singletons such as ST156 in pneumonia, and ST161 and ST396 in AOM. In contrast, eight STs were identified in meningitis, of which seven were genotypically closely related, while ST54 was the most frequent (62.9 %), unlike in the MLST database registrations, where ST6 predominated.

Conclusion. Non-typeable H. influenzae (NTHi), mostly derived from pneumonia and AOM, were genetically diverse, in contrast to the predominance of H. influenzae type b (Hib) among meningitis cases. The associations between certain STs and β-lactam resistance among NTHi were confirmed.

Purpose. Pneumococcal virulence protein-based vaccines can provide serotype-independent protection against pneumococcal infections. Many studies, including clinical observational studies on Thomsen–Friedenrich antigen exposure and haemolytic uremic syndrome, defined the role of neuraminidases NanA, NanB and NanC in host-pneumococcus interaction. Since neuraminidases are major virulence proteins, they are potential targets for both vaccines and small molecule inhibitors. Here we explored the utility of three neuraminidases as protein vaccine antigens to generate neutralizing antibodies and to increase survival following pneumococcal infections.

Methodology. Rabbits and mice were immunized subcutaneously with enzymatically active recombinant NanA, NanB and NanC as individual or a combination of the three neuraminidases. Antisera titres were determined by ELISA. Neuraminidase activity inhibition by antiserum was tested by peanut lectin and flow cytometry. Clinical isolates with serotype 3, 6B, 14, 15B, 19A and 23F were used to infect immunized mice by tail vein injection.

Results/Key findings. Presence of high levels of IgG antibodies in antisera against NanA, NanB and NanC indicates that all of the three neuraminidases are immunogenic vaccine antigens. To generate potent NanA neutralizing antibodies, both lectin and catalytic domains are essential, whereas for NanB and NanC a single lectin domain is sufficient. Immunization with triple neuraminidases increased the survival of mice when intravenously challenged with clinical isolates of serotype 3 (40 %), 6B (60 %), 15B (60 %), 19A (40 %) and 23F (30 %).

Conclusion. We recommend the inclusion of three pneumococcal neuraminidases in future protein vaccine formulations to prevent invasive pneumococcal infection caused by various serotypes.

Purpose. In enterohaemorrhagic Escherichia coli (EHEC), stx1 or stx2 genes encode Shiga toxin (Stx1 or Stx2, respectively) and are carried by prophages. The production and release of both stx phages and toxin occur upon initiation of the phage lytic cycle. Phages can further disseminate stx genes by infecting naïve bacteria in the intestine. Here, the effect of RNase E deficiency on these two virulence traits was investigated.

Methodology. Cultures of the EHEC strains TEA028-rne containing low versus normal RNase E levels or the parental strain (TEA028) were treated with mitomycin C (MMC) to induce the phage lytic cycle. Phages and Stx2 titres were quantified by the double-agar assay and the receptor ELISA technique, respectively.

Results. RNase E deficiency in MMC-treated cells significantly reduced the yield of infectious stx2 phages. Delayed cell lysis and the appearance of encapsidated phage DNA copies suggest a slow onset of the lytic cycle. However, these observations do not entirely explain the decrease of phage yields. stx1 phages were not detected under normal or deficient RNase E levels. After an initial delay, high levels of toxin were finally produced in MMC-treated cultures.

Conclusion. RNase E scarcity reduces stx2 phage production but not toxin. Normal concentrations of RNase E are likely required for correct phage morphogenesis. Our future work will address the mechanism of RNase E action on phage morphogenesis.