- Volume 63, Issue 3, 2014

Methyl-accepting chemotaxis proteins (MCPs), also termed transducer-like proteins (Tlps), serve as sensors in bacterial chemotactic signalling, and detect attractants and promote bacterial movement towards suitable sites for colonization. Campylobacter jejuni is a leading cause of human enteritis, but the mechanisms responsible for bacterial chemotaxis and early colonization in the jejunum of hosts are poorly understood. In the present study, we identified several types of bile and sodium deoxycholate (SDC) acting as chemotactic attractants of C. jejuni strain NCTC 11168-O in vitro, in which SDC was the most efficient chemoattractant. In mice with bile duct ligation, the wild-type strain displayed a markedly attenuated ability for colonization. Blockage of Tlp3 or Tlp4 protein with antibody or disruption of the tlp3 or tlp4 gene (Δtlp3 or Δtlp4) caused a significant inhibition of SDC-induced chemotaxis and attenuation for colonization on jejunal mucosa in mice of the bacterium. Disruption of both the genes (Δtlp3/Δtlp4) resulted in the absence of bacterial chemotaxis and colonization, while the tlp-gene-complemented mutants (CΔtlp3 and CΔtlp4) reacquired these abilities. The results indicate that SDC is an effective chemoattractant for C. jejuni, and Tlp3 and Tlp4 are the SDC-specific sensor proteins responsible for the bacterial chemoattraction.

A growing number of β-lactamases have been reported in Pseudomonas aeruginosa clinical isolates. The aim of this study was to investigate the diversity of β-lactamases in the collection of 51 ceftazidime-resistant P. aeruginosa clinical isolates in four hospitals of southern China. Among these isolates, variable degrees of resistance to other β-lactam and non-β-lactam agents were observed. Pulsed-field gel electrophoresis (PFGE) revealed a high degree of clonality with five main genotypes. Of the 51 isolates tested, 35 (68.6 %) were identified as extended-spectrum β-lactamase (ESBL) producers, with 35 producing PER-1, 1 CTX-M-3, 7 CTX-M-15 and 1 CTX-M-14. Most (82.9 %, 29/35) PER-1-producing isolates were collected from two hospitals between January and April in 2008 and belonged to the same PFGE pattern (pattern B) with similar antibiogram and β-lactamase profiles, which suggested an outbreak of this clone at the time. The prevalence of CTX-M-type ESBL (17.6 %, 9/51) was unexpectedly high. One isolate was identified as producing VIM-2. Furthermore, we also reported an occurrence of a novel OXA-10 variant, OXA-246, in 14 P. aeruginosa isolates. In addition, AmpC overproduction was found to be the β-lactamase-mediated mechanism responsible for ceftazidime resistance in 6 isolates (11.8 %). Our results revealed an overall diversity of β-lactamases and outbreak of a PER-1-producing clone among ceftazidime-resistant P. aeruginosa in southern China.

Chlamydia psittaci and atypical Chlamydiaceae infections are (re)-emerging in chickens. We therefore examined the prevalence of C. psittaci, atypical Chlamydiaceae and their zoonotic transmission on 19 Belgian chicken farms. Atypical Chlamydiaceae were not detected in chickens but 18 out of 19 farms were positive for C. psittaci by culture and PCR. C. psittaci ompA genotypes A and D were discovered. None of the examined humans (n = 31) was infected with atypical Chlamydiaceae, but 29 (93.5 %) of them were positive for C. psittaci by culture and PCR. Genotypes A, D and a mixed infection with genotypes C and D were found. Humans (n = 2) working at the C. psittaci-negative farm never had respiratory complaints, while 25 out of 29 positive farmers (86.2 %) reported yearly medical complaints potentially related to psittacosis. Four of them currently experienced respiratory disease and one of them was being treated with antibiotics. Four farmers (12.5 %) mentioned that they had pneumonia after starting to keep chickens. Occupational physicians should be aware of emerging Chlamydiaceae infections in chickens.

Human enterovirus 68 (HEV-68) is an enterovirus associated with respiratory illness. In China, no information about HEV-68 is available for children yet. This study aimed to investigate the presence of HEV-68 in mainland China between 2009 and 2012 and to explore the migration events of HEV-68 across the world. Among 1565 samples tested from children, 41 (2.6 %) were positive for HEV and 223 (14.3 %) for human rhinovirus (HRV). Seven (17.1 %) of 41 HEVs were HEV-68. Two HEV-68- and five HRV-positive samples were detected in 585 adult samples. HEV-68 is the predominant type of enterovirus in children with acute respiratory tract infection (ARTI), followed by HEV-71 and coxsackievirus A6. Three HEV-68-infected children presented with severe pneumonia and one presented with a severe asthma attack. The viruses were attributed to two novel distinct sublineages of HEV-68 based on phylogenetic analysis of partial VP1 gene sequences. Migration events analysis showed that the USA and the Netherlands were possible geographical sources of HEV-68, from where three strains migrated to China. In conclusion, HEV-68 may play a predominant role among the enteroviruses associated with ARTI in children. Additional surveillance is needed to clarify the reason why HEV-68 causes such a wide spectrum of disease, from asymptomatic to severe respiratory disease and even death.

Since 2007, Kenya has experienced an increase in cholera outbreaks characterized by a high fatality rate. In this study, we characterized 81 Vibrio cholerae isolates from diarrhoeal stool samples in Nyanza, a cholera-endemic lake region of Kenya, for virulence properties, clonality and antibiotic susceptibility. Eighty of these isolates were V. cholerae O1 El Tor variants carrying the classical ctxB gene sequence, while one isolate was V. cholerae non-O1/O139. All of the El Tor variants were of clonal origin, as revealed by PFGE, and were susceptible to ampicillin, tetracycline, ciprofloxacin, fosfomycin, kanamycin and norfloxacin. However, the isolates showed resistance to sulfamethoxazole/trimethoprim and streptomycin, and intermediate resistance to nalidixic acid, chloramphenicol and imipenem. The non-O1/O139 isolate carried the cholix toxin II gene (chxA II) and was susceptible to all antimicrobials tested except ampicillin. We propose that an El Tor variant clone caused the Nyanza cholera outbreak of 2007–2008.

There are sparse data concerning sex- and age-specific characteristics of community-acquired bacteraemia (CAB) and hospital-acquired bacteraemia (HAB). Between January 2008 and December 2012, we identified 2956 bacteraemia cases, which we classified as CAB, HAB or healthcare-associated bacteraemia (HCAB). Almost half of the pathogens were Escherichia coli in CAB patients. By contrast, Staphylococcus aureus was most frequent (16.2 %) in HAB patients. HCAB showed mixed features of CAB and HAB. In CAB, E. coli was significantly more abundant in females than in males (56.9  vs 24.3 %, respectively). This trend was most striking in young adults (20–39 years) (77.2 % in females vs 11.4 % in males). HAB cases showed greater heterogeneity in their associated pathogens. The extended-spectrum β-lactamase-positive rates of E. coli and Klebsiella pneumoniae, respectively, were 31.3 and 33.8 % in HAB and 8.8 and 8.4 % in CAB. The non-susceptibility rates of S. aureus to oxacillin were 37.4 % in CAB and 73.0 % in HAB. In conclusion, CAB and HAB showed different distributions of micro-organisms, and these distributions also differed with patient age and sex. In addition, antimicrobial susceptibility needs to be monitored separately.

Although 16S rRNA gene (rDNA) sequencing is the gold standard for categorizing bacteria or characterizing microbial communities its clinical utility is limited by bias in metagenomic studies, in either the experiments or the data analyses. To evaluate the efficiency of current metagenomic methods, we sequenced seven simulated samples of ten bacterial species mixed at different concentrations. The V3 region of 16S rDNA was targeted and used to determine the distribution of bacterial species. The number of target sequences in individual simulated samples was in the range 1–1000 to provide a better reflection of natural microbial communities. However, for a given bacterial species present in the same proportion but at different concentrations, the observed percentage of 16S rDNAs was similar, except at very low concentrations that cannot be detected by real-time PCR. These results confirmed that the comparative microbiome in a sample characterized by 16S rDNA sequencing is sufficient to detect not only potential infectious pathogens, but also the relative proportion of 16S rDNA in the sample.

Current diagnostics for community-acquired pneumonia (CAP) include testing for a wide range of pathogens, which is costly and not always informative. We compared clinical and laboratory parameters of patients with CAP caused by different groups of pathogens to evaluate the potential for targeted diagnostics and directed treatment. In a prospective study, conducted between April 2008 and April 2009, adult patients with CAP were tested for the presence of a broad range of possible respiratory pathogens using bacterial cultures, PCR, urinary antigen testing and serology. Of 408 patients with CAP, pathogens were detected in 263 patients (64.5 %). Streptococcus pneumoniae and influenza A virus were the most frequently identified bacterial and viral pathogens, respectively. Age had a significant effect on the prediction of aetiology (P = 0.054), with an increase in the relative contribution of viruses with advancing age. Multivariate analyses further showed that the presence of cough increased the likelihood of detecting a viral pathogen [odds ratio (OR) 5.536, 95 % confidence interval (CI) 2.130–14.390], the presence of immunodeficiency decreased the likelihood of detecting a bacterial pathogen (OR 0.595, 95 % CI 0.246–1.437) and an increase in pneumonia severity index score increased the likelihood of detecting a pathogen in general. Although several variables were independently associated with the detection of a pathogen group, substantial overlap meant there were no reliable clinical predictors to distinguish aetiologies. Therefore, testing for common respiratory pathogens is still necessary to optimize treatment.

Primary clarithromycin resistance is the main factor affecting the efficacy of Helicobacter pylori therapy. This study aimed: (i) to assess the concordance between phenotypic (culture) and genotypic (real-time PCR) tests in resistant strains; (ii) to search, in the case of disagreement between the methods, for point mutations other than those reported as the most frequent in Europe; and (iii) to compare the MICs associated with the single point mutations. In order to perform real-time PCR, we retrieved biopsies from patients in whom H. pylori infection was successful diagnosed by bacterial culture and clarithromycin resistance was assessed using the Etest. Only patients who had never been previously treated, and with H. pylori strains that were either resistant exclusively to clarithromycin or without any resistance, were included. Biopsies from 82 infected patients were analysed, including 42 strains that were clarithromycin resistant and 40 that were clarithromycin susceptible on culture. On genotypic analysis, at least one of the three most frequently reported point mutations (A2142C, A2142G and A2143G) was detected in only 23 cases (54.8 %), with a concordance between the two methods of 0.67. Novel point mutations (A2115G, G2141A and A2144T) were detected in a further 14 out of 19 discordant cases, increasing the resistance detection rate of PCR to 88 % (P<0.001; odds ratio 6.1, 95 % confidence interval 2−18.6) and the concordance to 0.81. No significant differences in MIC values among different point mutations were observed. This study suggests that: (i) the prevalence of the usually reported point mutations may be decreasing, with a concomitant emergence of new mutations; (ii) PCR-based methods should search for at least six point mutations to achieve good accuracy in detecting clarithromycin resistance; and (iii) none of the tested point mutations is associated with significantly higher MIC values than the others.

Data on the occurrence and epidemiology of Aspergillus spp. in laying hens farms are scant. With the aims of determining levels of airborne contamination in laying hen farms and evaluating the potential risk of infection for workers and animals, 57 air samples from 19 sheds (Group I), 69 from faeces (Group II), 19 from poultry feedstuffs (Group III) and 60 from three anatomical sites (i.e. nostrils, pharynx, ears) of 20 farm workers (Group IV) were cultured. The Aspergillus spp. prevalence in samples ranged from 31.6 % (Group III) to 55.5 % (Group IV), whereas the highest conidia concentration was retrieved in Group II (1.2×104 c.f.u. g−1) and in Group III (1.9×103 c.f.u. g−1). The mean concentration of airborne Aspergillus spp. conidia was 70 c.f.u. m−3 with Aspergillus fumigatus (27.3 %) being the most frequently detected species, followed by Aspergillus flavus (6.3 %). These Aspergillus spp. were also isolated from human nostrils (40 %) and ears (35 %) (P<0.05) (Group IV). No clinical aspergillosis was diagnosed in hens. The results demonstrate a relationship between the environmental contamination in hen farms and presence of Aspergillus spp. on animals and humans. Even if the concentration of airborne Aspergillus spp. conidia (i.e. 70 c.f.u. m−3) herein detected does not trigger clinical disease in hens, it causes human colonization. Correct management of hen farms is necessary to control environmental contamination by Aspergillus spp., and could lead to a significant reduction of animal and human colonization.

Neonatal sepsis due to carbapenem-resistant bacteria is difficult to treat due to limited therapeutic options. The detection of the new carbapenemase New Delhi metallo-β-lactamase-1 (NDM-1) from neonates has further complicated the situation (Roy et al., 2011a). The potent metallo-β-lactamase NDM-1 efficiently hydrolyses all classes of β-lactam antibiotics (penicillins, cephalosporins and carbapenems) and is also associated with multiple determinants that enable the bacteria to become resistant to other antibiotic classes ( Nordmann et al., 2011 ). In the presence of NDM-1 other β-lactamases may go unobserved because of the spectrum of activity of NDM-1 against all β-lactam antibiotics. Thus, under the canopy of the NDM-1 these β-lactamases also get the opportunity to spread. This communication reports association of two novel β-lactamases, SHV-type β-lactamase (SHV-167) and AmpC-type β-lactamase (ACT-16), in two NDM-1-carrying Enterobacteriaceae isolated from the blood of two septicaemic neonates admitted to a neonatal intensive care unit.