- Volume 63, Issue 12, 2014

Antibiotic susceptibility testing with the BD Phoenix system on bacterial cell pellets generated from blood culture broths using the Bruker MALDI Sepsityper kit was evaluated. Seventy-six Gram-negative isolates, including 12 with defined multi-resistant phenotypes, had antibiotic susceptibility testing (AST) performed by Phoenix on the cell pellet in parallel with conventional methods. In total, 1414/1444 (97.9 %) of susceptibility tests were concordant, with only 1 (0.07 %) very major error. This novel method has the potential to reduce the turnaround time for AST results by up to a day for Gram-negative bacteraemias.

Enteropathogenic Escherichia coli (EPEC) are a major cause of infant diarrhoea in developing countries and a significant public health issue in industrialized countries. Currently there are no simple tests available for the diagnosis of EPEC. Serology of O-antigens is widely used routinely in many laboratories throughout the world, even though it has been known for many years to be an unreliable indicator of EPEC virulence. We have developed a simple, low-cost immunodiagnostic test based on the EspA filament, an essential virulence factor of EPEC and the related enterohaemorrhagic E. coli (EHEC). Using recombinant proteins of the five major variants of EspA as immunogens, we raised a panel of three monoclonal antibodies in mice that detects all variants of the native target in bacterial cultures. The antibodies proved suitable for application in sandwich-type assays, including ELISA and lateral flow immunoassays (LFI). Prototypes for both assays were specific for EPEC and EHEC strains when tested against a panel of control micro-organisms. We have also developed a simple, affordable culture medium, A/E medium, which optimizes expression of EspA allowing improved sensitivity of detection compared with standard Dulbecco’s modified Eagle’s medium. Together these reagents form the basis of robust, informative tests for EPEC for use especially in developing countries but also for routine screening in any clinical laboratory.

Hypervirulent Klebsiella pneumoniae isolates of capsular serotype K2 (hvKP-K2) that cause community-acquired invasive infections represent several unrelated clones, which all belong to phylogenetic group KpI. These clones can be recognized using multilocus sequence typing and genomic analyses, but no rapid method currently exists to differentiate them. In this work, a multiplex PCR assay was developed to identify three hvKP-K2 groups: (i) sequence type (ST)86; (ii) ST380 and ST679 (i.e. clonal group 380); and (iii) ST65 and ST375. A specific genetic marker, Kp50233, allowing K. pneumoniae sensu stricto (corresponding to phylogroup KpI) to be distinguished from closely related species, was included in the assay. This PCR assay will be useful in better defining the epidemiology and clinical features of emerging virulent K. pneumoniae clones.

Stenotrophomonas maltophilia is an opportunist multidrug-resistant pathogen that causes a wide range of nosocomial infections. Various cystic fibrosis (CF) centres have reported an increasing prevalence of S. maltophilia colonization/infection among patients with this disease. The purpose of this study was to assess specific fingerprints of S. maltophilia isolates from CF patients (n = 71) by investigating fatty acid methyl esters (FAMEs) through gas chromatography (GC) and highly abundant proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and to compare them with isolates obtained from intensive care unit (ICU) patients (n = 20) and the environment (n = 11). Principal component analysis (PCA) of GC-FAME patterns did not reveal a clustering corresponding to distinct CF, ICU or environmental types. Based on the peak area index, it was observed that S. maltophilia isolates from CF patients produced significantly higher amounts of fatty acids in comparison with ICU patients and the environmental isolates. Hierarchical cluster analysis (HCA) based on the MALDI-TOF MS peak profiles of S. maltophilia revealed the presence of five large clusters, suggesting a high phenotypic diversity. Although HCA of MALDI-TOF mass spectra did not result in distinct clusters predominantly composed of CF isolates, PCA revealed the presence of a distinct cluster composed of S. maltophilia isolates from CF patients. Our data suggest that S. maltophilia colonizing CF patients tend to modify not only their fatty acid patterns but also their protein patterns as a response to adaptation in the unfavourable environment of the CF lung.

Non-invasive diagnosis of Helicobacter pylori infection is important not only for screening of infection but also for epidemiological studies. Stool antigen tests are non-invasive and are convenient to identify H. pylori infection, particularly in children. We evaluated the stool antigen test, which uses a mAb for native catalase of H. pylori developed in Japan. A total of 151 stool samples were collected from participants (52 children and 99 adults) of the Sasayama Cohort Study and stored between −30 and −80 °C. The stool antigen test used was Testmate pylori antigen (TPAg), and was performed according to the manufacturer’s instructions. Furthermore, we conducted a quantitative real-time PCR test and compared the PCR results with those of the TPAg test. When compared with the results in real-time PCR, the sensitivity of TPAg was 89.5 % overall, 82.7 % for children and 92.4 % for adults, and the specificity was 100 %. The accuracy was 93.4 % overall, 90.4 % for children and 94.9 % for adults, and there was no significant difference in the accuracy of TPAg between children and adults. Five of 28 children (18 %) and five of 38 adults (13 %) were PCR positive with negative TPAg results. Four of five children with positive PCR and negative TPAg results were given a 13C-urea breath test and all four children tested negative. No significant correlation was observed between the TPAg results and DNA numbers of H. pylori in faeces among children or adults. A stool antigen test (TPAg) using a mAb for native catalase is useful for diagnosis of H. pylori in children and adults. Additionally, this test has particularly high specificity.

This study was conducted as part of the Argentinean Influenza and other Respiratory Viruses Surveillance Network, in the context of the Global Influenza Surveillance carried out by the World Health Organization (WHO). The objective was to study the activity and the antigenic and genomic characteristics of circulating viruses for three consecutive seasons (2010, 2011 and 2012) in order to investigate the emergence of influenza viral variants. During the study period, influenza virus circulation was detected from January to December. Influenza A and B, and all current subtypes of human influenza viruses, were present each year. Throughout the 2010 post-pandemic season, influenza A(H1N1)pdm09, unexpectedly, almost disappeared. The haemagglutinin (HA) of the A(H1N1)pdm09 viruses studied were segregated in a different genetic group to those identified during the 2009 pandemic, although they were still antigenically closely related to the vaccine strain A/California/07/2009. Influenza A(H3N2) viruses were the predominant strains circulating during the 2011 season, accounting for nearly 76 % of influenza viruses identified. That year, all HA sequences of the A(H3N2) viruses tested fell into the A/Victoria/208/2009 genetic clade, but remained antigenically related to A/Perth/16/2009 (reference vaccine recommended for this three-year period). A(H3N2) viruses isolated in 2012 were antigenically closely related to A/Victoria/361/2011, recommended by the WHO as the H3 component for the 2013 Southern Hemisphere formulation. B viruses belonging to the B/Victoria lineage circulated in 2010. A mixed circulation of viral variants of both B/Victoria and B/Yamagata lineages was detected in 2012, with the former being predominant. A(H1N1)pdm09 viruses remained antigenically closely related to the vaccine virus A/California/7/2009; A(H3N2) viruses continually evolved into new antigenic clusters and both B lineages, B/Victoria/2/87-like and B/Yamagata/16/88-like viruses, were observed during the study period. The virological surveillance showed that the majority of the circulating strains during the study period were antigenically related to the corresponding Southern Hemisphere vaccine strains except for the 2012 A(H3N2) viruses.

Carbapenems are the last-resort antibiotics for the treatment of infections caused by multidrug-resistant Gram-negative bacilli. Klebsiella pneumoniae carbapenemase (KPC) hydrolyses β-lactam antibiotics including the carbapenems. KPCs have been detected in Enterobacteriaceae and Pseudomonas aeruginosa isolates worldwide associated with transposon Tn4401 commonly located in plasmids. Acinetobacter baumannii has become an important multidrug-resistant nosocomial pathogen capable of hydrolysing the carbapenem antibiotics. KPC-producing A. baumannii has so far only been reported in Puerto Rico. During a surveillance study, four KPC-producing A. baumannii with identical pulse type were identified in a single institution. The objectives of this study were to characterize the KPC genetic background and the allelic diversity of one of the isolates. Next-generation sequencing and multilocus sequence typing (MLST) were performed. Molecular characterization of the isolate demonstrated bla KPC in Tn4401b located in the bacterial chromosome within a 26.5 kb DNA fragment, which included a KQ-like element (18.9 kb) very similar to that described previously in a K. pneumoniae plasmid and a 7.6 kb DNA fragment with 98 % homology to a putative plasmid from Yersinia pestis strain PY-95. Our data suggested that the 26.5 kb DNA fragment harbouring bla KPC was integrated in the chromosome by a transposition event mediated by the transposase of ISEcp1 found in the KQ-like element. MLST showed a novel sequence type, ST250. To our knowledge, this is the first report of the identification of the genetic background of bla KPC in A. baumannii.

Antibiotic-resistant bacteria have emerged from the widespread use of antibiotics worldwide and have prompted the search for new sources of antimicrobial substances. Pinus spp. contain several bioactive compounds consisting mainly of terpenes, terpenoids and some other aromatic and aliphatic constituents. These compounds exert important biological effects, and pine oils have found wide application in the industry. In the present study, we have evaluated the potential activity of the resin-oil of Pinus elliottii and its major compound dehydroabietic acid (DA) against multiresistant bacteria by MIC, minimum bactericidal concentration and time-kill assays. The MIC of the resin-oil of P. elliottii varied between 25 and 100 µg ml−1. As for DA, the MIC and minimum bactericidal concentration varied between 6.25 and 50 and between 6.25 and 100 µg ml−1, respectively. The time-kill assay conducted with DA at 6.25 µg ml−1 evidenced bactericidal activity against Staphylococcus epidermidis (American Type Culture Collection 14990) within 24 h. On the basis of these results, the resin-oil of P. elliottii and its major compound DA play an important part in the search for novel sources of agents that can act against multiresistant bacteria.

This study investigated the molecular epidemiology of Acinetobacter baumannii in the University of Debrecen in relation to antibiotic consumption. Overall and ward-specific antibiotic consumption was measured by the number of defined daily doses (DDD) per 100 bed-days between 2002 and 2012. Consumption was analysed against the number of A. baumannii positive patients per 100 bed-days, number of isolates per positive sample, and proportion of carbapenem resistant A. baumannii, using time-series analysis. Altogether 160 A. baumannii isolates from different wards were collected and analysed. Carbapenemase genes blaOXA-23-like , blaOXA-24-like , blaOXA-48-like , blaOXA-51-like , blaOXA-58-like and integrons were sought by PCR. Relatedness of isolates was assessed by PFGE. Prevalence and carbapenem resistance of A. baumannii were statistically associated with carbapenem consumption. Prevalence data followed carbapenem usage with three quarterly lags (r = 0.51–0.53, P<0.001), and meropenem and ertapenem, but not imipenem usage, affected prevalence. Colistin usage, in turn, lagged behind prevalence with one lag (r = 0.68–0.70, P<0.001). Six clusters were identified; the neurology ward with the lowest carbapenem consumption was associated with the carbapenem-susceptible cluster, as well as with the carbapenem-susceptible isolates in the cluster with variable susceptibility. Wards with high carbapenem usage almost exclusively harboured isolates from carbapenem-resistant clusters. All clusters were dominated by isolates of one or two wards, but most wards were represented in multiple clusters. Increases in prevalence and carbapenem resistance of A. baumannii were associated with usage of meropenem and ertapenem but not of imipenem, which led to the spread of multiple clones in the University.

The major risk factor for Clostridium difficile infection (CDI) is the use of antibiotics owing to the disruption of the equilibrium of the host gut microbiota. To preserve the beneficial resident probiotic bacteria during infection treatment, the use of molecules with selective antibacterial activity enhances the efficacy by selectively removing C. difficile. One of them is the plant alkaloid 8-hydroxyquinoline (8HQ), which has been shown to selectively inhibit clostridia without repressing bifidobacteria. Selective antimicrobial activity is generally tested by culture techniques of individual bacterial strains. However, the main limitation of these techniques is the inability to describe differential growth dynamics of more bacterial strains in co-culture within the same experiment. In the present study, we combined fluorescent in situ hybridization and flow cytometry to describe the changes in active and non-active cells of a mixed culture formed by the opportunistic pathogen C. difficile CECT 531 and the beneficial Bifidobacterium longum subsp. longum CCMDMND BL1 after exposure to 8HQ. It was observed that without 8HQ, the proportion of both strains was almost equal, oscillating between 22.7 and 77.9 % during a time lapse of 12 h, whereas with 8HQ the proportion of active C. difficile decreased after 4 h, and persisted only between 8.8 and 17.5 %. In contrast, bifidobacterial growth was not disturbed by 8HQ. The results of this study showed the selective inhibitory effect of 8HQ on clostridial and bifidobacterial growth dynamics, and the potential of this compound for the development of selective agents to control CDIs.

The rates of multidrug-resistant, extensively drug-resistant and pandrug-resistant isolates amongst non-fermenting Gram-negative bacilli, particularly Pseudomonas aeruginosa, have risen worldwide. The clinical consequence of resistance and the impact of adverse treatment on the outcome of patients with P. aeruginosa bacteraemia remain unclear. To better understand the predictors of mortality, the clinical consequence of resistance and the impact of inappropriate therapy on patient outcomes, we analysed the first episode of P. aeruginosa bacteraemia in patients from a Brazilian tertiary-care hospital during the period from May 2009 to August 2011. Antimicrobial susceptibility testing was conducted; phenotypic detection of metallo-β-lactamase (MBL) and PCR of MBL genes were performed on carbapenem-resistant strains. Amongst the 120 P. aeruginosa isolates, 45.8 % were resistant to carbapenem and 36 strains were tested for MBL detection. A total of 30 % were phenotypically positive and, of these, 77.8 % expressed an MBL gene, bla SPM-1 (57 %) and bla VIM-type (43 %). The resistance rates to ceftazidime, cefepime, piperacillin/tazobactam, carbapenem, fluoroquinolone and aminoglycoside were 55, 42.5, 35, 45.8, 44 and 44 %, respectively. Previous antibiotic use, length of a hospital stay ≥30 days prior to P. aeruginosa, haemodialysis, tracheostomy, pulmonary source of bacteraemia and Intensive Care Unit admission were common independent risk factors for antimicrobial resistance. Cefepime resistance, multidrug resistance and extensive drug resistance were independently associated with inappropriate therapy, which was an important predictor of mortality, being synergistic with the severity of the underlying disease.

We retrospectively analysed the incidence rate of reported cases of pertussis in Barcelona during 2009–2012 according to age, sex, type of medical centre and vaccination status. We included 748 confirmed or suspected cases, 613 (82.0 %) of which were confirmed by laboratory testing and the remaining 135 (18.0 %) by epidemiological evidence. The highest reported incidence of pertussis was amongst <1 year olds [96.1 per 100 000 person-years, 95 % confidence interval (CI): 84.3–109.1]. The majority of confirmed and suspected cases were reported in 2011 and 2012, and the total incidence (confirmed or suspected) was 6.3 (95 % CI: 5.6–6.9) and 4.2 (95 % CI: 3.6–4.7) per 100 000 person-years, respectively. Incidence increased significantly (P = 0.001) in 2011–2012 compared with 2009. Most confirmed cases occurred in children <1 year old (87.9 %). Cases were confirmed by real-time (RT)-PCR (87.5 %; 95 % CI: 81.3–87.6) and bacterial culture (13.7 %; 95 % CI: 11.0–17.1). We recommend performing RT-PCR in suspected cases with no epidemiological link to a confirmed case.

During 2000–2004, 13 Shigella strains that were untypable by commercially available antisera were isolated from children <5 years of age with acute diarrhoea in Kolkata. These strains were subsequently identified as Shigella dysenteriae provisional serovar 204/96 (n = 3), Shigella dysenteriae provisional serovar E23507 (n = 1), Shigella dysenteriae provisional serovar I9809-73 (n = 1), Shigella dysenteriae provisional serovar 93-119 (n = 1), Shigella flexneri provisional serovar 88-893 (n = 6) and Shigella boydii provisional serovar E16553 (n = 1). In this study, characterization of those provisional serovars of Shigella was performed with respect to their antimicrobial resistance, plasmids, virulence genes and PFGE profiles. The drug resistant strains (n = 10) of Shigella identified in this study possessed various antibiotic resistance genetic markers like catA (for chloramphenicol resistance); tetA and tetB (for tetracycline resistance); dfrA1 and sul2 (for co-trimoxazole resistance); aadA1, strA and strB (for streptomycin resistance) and blaOXA-1 (for ampicillin resistance). Class 1 and/or class 2 integrons were present in eight resistant strains. Three study strains were pan-susceptible. A single mutation in the gyrA gene (serine to leucine at codon 83) was present in four quinolone resistant strains. The virulence gene ipaH (invasion plasmid antigen H) was uniformly present in all strains in this study, but the stx (Shiga toxin) and set1 (Shigella enterotoxin 1) genes were absent. Other virulence genes like ial (invasion associated locus) and sen (Shigella enterotoxin 2) were occasionally present. A large plasmid of 212 kb and of incompatibility type IncFIIA was present in the majority of the strains (n = 10) and diversity was noticed in the smaller plasmid profiles of these strains even within the same provisional serovars. PFGE profile analysis showed the presence of multiple unrelated clones among the isolates of provisional Shigella serovars. To the best of our knowledge, this is the first report on the phenotypic and molecular characterization of provisional serovars of Shigella isolates from Kolkata, India.

The role of Streptococcus pneumoniae in cystic fibrosis (CF) is poorly understood. The pneumococcal population has changed over time after the introduction of the heptavalent conjugate vaccine (PCV7) and, more recently, the 13-valent conjugate vaccine (PCV13). Although serotypes and clones causing invasive pneumococcal disease or colonizing healthy children have been extensively analysed, little is known so far on the serotypes and clones of pneumococci in CF patients. The aim of this work was to investigate serotypes, antibiotic susceptibilities, genotypes and biofilm production of CF pneumococcal isolates. Overall, 44 S. pneumoniae strains collected from 32 paediatric CF patients from January 2010 to May 2012 in a large Italian CF Centre were tested for antimicrobial susceptibility testing by Etest, serotyped by the Quellung reaction and genotyped by a combination of different molecular typing methods, including pbp gene restriction profiling, pspA restriction profiling and sequencing, PFGE and multilocus sequence typing. Biofilm production by pneumococcal strains was also assessed. Penicillin non-susceptibility was 16 %. High resistance rates (>56 %) were observed for erythromycin, clindamycin and tetracycline. The most frequent serotype recovered was serotype 3 (31.8 %). The coverage of PCV7 and PCV13 was 6.8 and 47.7 %, respectively. More than 80 % of CF strains belonged to Pneumococcal Molecular Epidemiology Network (PMEN) reference clones, the most common being Netherlands3-ST180 (28.2 %), and Greece21-30/ST193 (15.4 %). All strains produced biofilm in vitro, although with large variability in biofilm formation efficiency. No correlation was found between biofilm levels and serotype, clone or antibiotic resistance. The high isolation rate of antibiotic-resistant serotype 3 pneumococci from CF patients suggests that PCV13 could increase protection from pneumococcal colonization and infection.

Poliomyelitis, a disease which can manifest as muscle paralysis, is caused by the poliovirus, which is a human enterovirus and member of the family Picornaviridae that usually transmits by the faecal–oral route. The viruses of the OPV (oral poliovirus attenuated-live vaccine) strains can mutate in the human intestine during replication and some of these mutations can lead to the recovery of serious neurovirulence. Informatics research of the poliovirus genome can be used to explain further the characteristics of this virus. In this study, sequences from 100 poliovirus isolates were acquired from GenBank. To determine the evolutionary relationship between the strains, we compared and analysed the sequences of the complete poliovirus genome and the VP1 region. The reconstructed phylogenetic trees for the complete sequences and the VP1 sequences were both divided into two branches, indicating that the genetic relationships of the whole poliovirus genome and the VP1 sequences are very similar. This branching indicates that the virulence and pathogenicity of poliomyelitis may be associated with the VP1 region. Sequence alignment of the VP1 region revealed numerous mutation sites in which mutation rates of >30 % were detected. In a group of strains recorded in the USA, mutation sites and mutation types were the same and this may be associated with their distribution in the evolutionary tree and their genetic relationship. In conclusion, the genetic evolutionary relationships of poliovirus isolate sequences are determined to a great extent by the VP1 protein, and poliovirus strains located on the same branch of the phylogenetic tree contain the same mutation spots and mutation types. Hence, the genetic characteristics of the VP1 region in the poliovirus genome should be analysed to identify the transmission route of poliovirus and provide the basis of viral immunity development.