- Volume 65, Issue 10, 2016

Serotyping forms the basis of all national and international surveillance networks for Salmonella. Public health microbiology is currently being transformed by high-throughput DNA sequencing, which opens the door to serovar determination using this powerful technique. Twenty-nine Salmonella isolates referred to the Public Health England between 1994 and 2004 for serovar identification were selected for this study, and they all presented with novel antigenic formulae. Results from a combination of traditional phenotypic and molecular assays were compared. Twenty-two isolates (76 %) were subsequently independently confirmed as new types; of these, 18 (82 %) were grouped as Salmonella enterica subspecies I, and four (18 %) were S. enterica subspecies II. In general, it is shown that there is concordance between the DNA sequence type and traditional phenotypic serotype, but it would be necessary to analyse a larger data set to confirm this. Traditional multilocus sequence typing (MLST) by Sanger sequencing also correlates to in silico whole-genome sequencing MLST. This permits the continuation of traditional serovar nomenclature alongside sequence type methods and enhances the ability to infer true phylogenetic relationships between isolates.

Dissemination of antibiotic resistance in Enterobacteriaceae mediated by AmpC β-lactamase, extended-spectrum β-lactamase (ESBL) and metallo-β-lactamase (MBL) is clinically significant. A simple and relatively quick method for the detection of these resistance phenotypes would greatly improve chemotherapeutic recommendation. This technology would provide valuable input in our surveillance of resistance on a global stage, particularly if the methodology could be applicable to resource-poor settings. A resazurin microtitre plate (RMP) assay incorporating cloxacillin, clavulanic acid and EDTA for the rapid phenotypic identification of AmpC, ESBL and MBL and the co-existence of β-lactamases has been developed. A total of 47 molecularly characterized Enterobacteriaceae clinical isolates producing AmpCs, ESBLs, co-producers of ESBL and AmpC, MBLs and co-producers of ESBL and MBL were phenotypically examined using the RMP assay. The ceftazidime- and cefotaxime-based RMP assays successfully detected all 16 AmpC, 14 ESBL and 9 MBL producers, 6 ESBL–AmpC co-producers and 2 ESBL–MBL co-producers without false-positive results. The ceftazidime-based assay was more reliable in detecting AmpC alone, while the cefotaxime-based assay performed better in identifying co-producers of ESBL and AmpC. There was no difference in the detection of ESBL and MBL producers. The findings of the present study suggest that use of the RMP assay with particular β-lactamase inhibitors explicitly detects three different β-lactamases, as well as co-existence of β-lactamases, within 6 h of initial isolation of the pathogen. This assay is applicable to carry out in any laboratory, is cost-effective and is easy to interpret. It could be implemented in screening patients and controlling infection and for surveillance purposes.

One hundred and twenty-six epidemiologically sequential, unrelated, carbapenem-resistant Acinetobacter baumannii isolates from nine hospitals in six countries of South America were collected between July 2013 and June 2014. Genes coding for Ambler class D and B carbapenemases were sought by PCR. All isolates were typed using the 3-locus sequence typing and bla OXA-51-like sequence-based typing techniques. The bla OXA-23 gene was recovered in all the participating hospitals and in all the isolates of seven of nine medical centres. The bla OXA-72 gene was only recovered in the two medical centres from Guayaquil city, Ecuador. Trilocus sequence typing revealed the presence of sequence groups SG2, SG4 and SG5. bla OXA-51-like sequence-based typing revealed the presence of bla OXA-132, bla OXA-65, bla OXA-69 and bla OXA-64. Our results showed that the population of carbapenem-resistant A. baumannii in South America was principally associated with ST79, ST25 and ST15 (92 %) and harboured the bla OXA-23 gene mainly. CC2 was not detected.

Diabetic patients frequently develop diabetic foot ulcers (DFUs), particularly those patients vulnerable to Staphylococcus aureus opportunistic infections. It is urgent to find new treatments for bacterial infections. The antimicrobial peptide (AMP) nisin is a potential candidate, mainly due to its broad spectrum of action against pathogens. Considering that AMP can be degraded or inactivated before reaching its target at therapeutic concentrations, it is mandatory to establish effective AMP delivery systems, with the natural polysaccharide guar gum being one of the most promising. We analysed the antimicrobial potential of nisin against 23 S. aureus DFU biofilm-producing isolates. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) were determined for nisin diluted in HCl and incorporated in guar gum gel. Statistical analysis was performed using the Wilcoxon matched-pair test. Nisin was effective against all isolates, including some multidrug-resistant clinical isolates, independent of whether it is incorporated in guar gum. While differences among MIC, MBC and MBIC values were observed for HCl- and guar gum- nisin, no significant differences were found between MBEC values. Inhibitory activity of both systems seems to differ only twofold, which does not compromise guar gum gel efficiency as a delivery system. Our results highlight the potential of nisin as a substitute for or complementary therapy to current antibiotics used for treating DFU infections, which is extremely relevant considering the increase in multidrug-resistant bacteria dissemination. The guar gum gel represents an alternative, practical and safe delivery system for AMPs, allowing the development of novel topical therapies as treatments for bacterial skin infections.

Crimean–Congo hemorrhagic fever (CCHF) is a life-threatening disease that develops as a result of infection by a member of the Nairovirus genus of the Bunyaviridae family, and its initial symptoms are not specific. In patients with severe clinical progression, in particular, the neutrophil rate is high, whereas lymphocyte and monocyte levels are low. A total of 149 patients, in whom the diagnosis was confirmed with reverse transcriptase PCR, were included in the study. In order to compare patient clinical progression severity, we divided the patients into two groups. For group 1, Çevik’s severity score was used. The patients who had a platelet/lymphocyte ratio (PLR) <41 constituted group 2. Of 149 patients, 20 (13.4 %) were determined as group 1 (Çevik’s classification) and 38 (25.5 %) were determined as group 2 (PLR <41). Of 11 deaths, 4 (36.4 %) patients were from group 1 and 7 (63.6 %) were from group 2. This is the first study to our knowledge to analyse the relationship between severity and PLR in patients with CCHF. PLR is a simple laboratory test that can aid in determining the prognosis of individuals with this disease.

Point-of-care testing for Mycoplasma pneumoniae infection may be ideal and useful because significant numbers of the cases will be seen as outpatients. Recently, a new immunochromatographic method (ICM) targeting M. pneumoniae ribosomal protein L7/L12 (RP-L7/L12) in pharyngeal swabs became available in Japan, although clinical data and basic information regarding efficacy and characterization of this ICM are limited. The present study examined the fate of M. pneumoniae RP-L7/L12 during in vitro growth and the correlation between M. pneumoniae concentration in clinical specimens and the sensitivity of the ICM test. The usefulness of the ICM was investigated in patients suspected of having M. pneumoniae pneumonia and upper respiratory tract infection (137 children and 39 adults). The limit of detection for the ICM test was 1.1×104 c.f.u. ml−1 of M. pneumoniae. Bacterial production of RP-L7/L12 correlated positively with the viable M. pneumoniae concentration in vitro; antigen was then degraded in culture broth, with an in vitro half-life of approximately 2 days. Five other Mycoplasma spp. and 14 representative respiratory pathogens were ICM assay negative at bacterial concentrations of 106 c.f.u. ml−1. The clinical sensitivity and specificity of the ICM assay were 57.1 % (20/35) and 92.2 % (130/141), respectively, in comparison with bacterial culture. Clinical specimens containing ≥106 c.f.u. ml−1 of M. pneumoniae burden were ICM positive in 13 of 18 cases (72.2 %). The ICM is a poorly sensitive but reasonably specific means for detecting M. pneumoniae infections.

The emergence of extensively drug-resistant (XDR) Klebsiella pneumoniae has become a major challenge worldwide. In this study, we characterized the phenotypes and genetic features of nine XDR K. pneumoniae isolates from an integrated hospital in Zhejiang province, China, from September to October 2014. These XDR K. pneumoniae possessed at least five resistance determinants which contribute to highly resistant to β-lactam, β-lactam/inhibitor combinations, aminoglycosides, quinolones, carbapenems, chloroamphenicol and fosfomycin. All isolates carried bla KPC-2, bla CTX-M-9, bla SHV-11 and rmtB, and several isolates also harboured bla TEM-1 and qnrS. Southern blot experiments confirmed that bla KPC-2, rmtB and bla CTX-M-9 were located on the same ~54.2 kb plasmid. Conjugative plasmids were obtained from all K. pneumoniae isolates, further proving the transferable characteristic of the resistance determinants. The OmpK36 sequences showed various deletions and insertions that indicated additional amino acid residues and a deleted phenotype of OmpK36. PFGE demonstrated that all the isolates belonged to the same genotype. Multilocus sequence typing was concordant with PFGE results and revealed that ST11 was the most predominant clone. Our study revealed a high incidence and endemic spread of XDR K. pneumoniae in the hospital. Thus, effective infection control measures should be adopted to monitor and control the spread of multidrug-resistant isolates.

We studied in vitro ceftaroline combinations against 61 meticillin-resistant Staphylococcus aureus isolates; 18 of them were also resistant to linezolid, using overlapping E-test method. Daptomycin–ceftaroline combination obtained lower fractional inhibitory concentration values, in comparison with those including vancomycin or linezolid against meticillin-resistant S. aureus (P<0.05). All meticillin- and linezolid-resistant S. aureus strains were resistant to ceftaroline; nevertheless, combinations with vancomycin or daptomycin showed higher synergy or addition rates than those with linezolid.

The study evaluated the in vitro impact of a series of aminophosphinic urease inhibitors on Proteus mirabilis. The group of compounds comprised structurally diverse analogues of diamidophosphate built on an N-C-P scaffold. The influence of urease inhibition on urea-splitting activity was assessed by whole-cell pH-static kinetic measurements. The potential to prevent struvite formation was determined by monitoring changes in pH and ionic composition of artificial urine medium during P. mirabilis growth. The most active compounds exhibited stronger positive effect on urine stability than the acknowledged inhibitor acetohydroxamic acid. The high anti-ureolytic and pH-stabilizing effect of urease inhibitors 4 and 14 was well correlated with their reported kinetic properties against pure urease from P. mirabilis (K i values of 0.62±0.09 and 0.202±0.057 µM, respectively, compared to 5.7±0.4 µM for acetohydroxamic acid). The effect of repressed ureolysis upon the viability of Proteus cells was studied using MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] metabolic efficiency assay and LIVE/DEAD fluorescent staining. Most of the compounds caused whole-cell dehydrogenase activity loss; four structures (1, 2, 4 and 14) reduced the culture viability by nearly 70 % at 1 mM concentration. Results of dual fluorescent staining suggested that besides urea-splitting prevention, the structures additionally exerted an outer-membrane-destabilizing effect.

Vibrio cholerae causes cholera outbreaks in endemic regions where the water quality and sanitation facilities remain poor. Apart from biotype and serotype changes, V. cholerae undergoes phase variation, which results in the generation of two morphologically different variants termed smooth and rugose. In this study, 12 rugose (R-VC) and 6 smooth (S-VC) V. cholerae O1 Ogawa isolates were identified in a cholera outbreak that occurred in Hyderabad, India. Antimicrobial susceptibility results showed that all the isolates were resistant to ampicillin, furazolidone and nalidixic acid. In addition, R-VC isolates were resistant to ciprofloxacin (92 %), streptomycin (92 %), erythromycin (83 %), trimethoprim-sulfamethoxazole (75 %) and tetracycline (75 %). Based on the ctxB gene analysis, all the isolates were identified as El Tor variant with mutation in two positions of ctxB, similar to the classical biotype. The R-VC isolates specifically showed excessive biofilm formation and were comparatively less motile. In addition, the majority of these isolates (~83 %) displayed random mutations in the hapR gene, which encodes haemagglutinin protease regulatory protein. In the PFGE analysis, R-VC and S-VC were placed in distinct clusters but remained clonally related. In the ribotyping analysis, all the R-VC isolates exhibited R-III pattern, which is a prevailing type among the current El Tor isolates. A hapR deletion mutant generated using an S-VC isolate expressed rugose phenotype. To our knowledge, this is the first report on the association of rugose V. cholerae O1 in a large cholera outbreak with extended antimicrobial resistance and random mutations in the haemagglutinin protease regulatory protein encoding gene (hapR).

This study aimed to determine the occurrence of Mannheimia haemolytica, Pasteurella multocida and Mycoplasma spp., in relation to clinical signs of respiratory disease. Tracheobronchial lavage samples were collected from 96 (healthy and unhealthy) cattle in the State of São Paulo, Brazil. Mycoplasma spp. (12.5 %) and Pasteurella multocida (15.50 %) were the most prevalent species. Bacillus sp., Staphylococcus sp., Escherichia coli, Klebsiella oxytoca, Pseudomonas aeruginosa and Klebsiella pneumoniae were also isolated. Mollicutes (70.83 %), Mycoplasma bovis (2.94 %) and Mycoplasma dispar (38.23 %) were identified using conventional PCR. Submassive sound on acoustic percussion of the thorax was associated with the absence of Mollicutes (P=0.025). Whistling (P=0.076) and coarse crackle (P=0.046) were associated with the absence of Mycoplasma dispar. Clear sound on acoustic percussion of the thorax was associated with the absence of Mycoplasma bovis (P=0.007). Coughing was associated with the presence of Pasteurella multocida [P=0.035; confidence interval (CI), 1.12–26.89], but its absence was associated with mucopurulent (P=0.0215; CI, 1.55–34.5) and mucoid nasal discharge (P=0.068; CI, 19–28.5), submassive sound (P=0.031; CI, 1.23–75.5), fine crackle (P=0.058; CI, 1.23–20.1) and coarse crackle (P=0.046; CI, 2.38–70.8). The high prevalence of Pasteurella multocida and Mycoplasma spp. in unhealthy calves increases the importance of these micro-organisms in the pathogenesis of respiratory diseases. This study increases the information about the role of Mycoplasma dispar in respiratory diseases. Differences in some species in relation to clinical signs can be applied as a presumptive diagnosis.

Applying self-sanitizing copper surfaces to commonly touched places within hospital facilities is an emerging strategy to prevent healthcare-associated infections. This is due to the fact that bacterial pathogens are rapidly killed on copper, a process termed contact killing. However, the mechanisms of contact killing are not fully understood, and the potential of bacterial pathogens to develop resistance has rarely been explored. Here, we hypothesize that bacteria are predominantly killed by a burst release of toxic copper ions, resulting from chemical reactions between bacterial cell surface components and metallic copper. To test this, we isolated and characterized small colony variants (SCVs) derived from Pseudomonas aeruginosa and Staphylococcus aureus. SCVs overproduce extracellular polymeric substances (EPS), which will enhance copper ion release, causing more rapid death on copper. Indeed, all 13 SCVs tested were more rapidly killed than wild-types on the surfaces of both pure copper and brass (63.5 % Cu). Next, using the non-pathogenic Pseudomonas fluorescens SBW25 as a model, we examined the roles of specific cell surface components in contact killing, including EPS, LPS, capsule, flagella and pili. We also subjected P. fluorescens SBW25 to daily serial passage of sub-lethal conditions on brass. After 100 transfers, there was a slight increase of survival rate, but ~97 % of cells can still be killed within 60 min on brass. Together, our data implicate that the rate of contact killing on copper is largely determined by the cell surface components, and bacteria have limited ability to evolve resistance to metallic copper.

In 2010, the 10-valent pneumococcal conjugate vaccine (PCV10) was introduced into the Brazilian childhood vaccination programme. Concerns have been raised that non-vaccine serotypes could increase in prevalence and reduce the benefits of vaccination; therefore, we examined non-PCV10 isolates recovered from meningitis during pre- (January 2008–May 2010) and post-vaccine (June 2010–December 2012) periods. Surveillance for pneumococcal meningitis was established at the Reference Hospital of Infectious Diseases in Salvador, Brazil. Serotypes were determined by multiplex PCR and/or Quellung reaction. Antimicrobial susceptibility testing was conducted by E-test and broth microdilution. Genotyping used PFGE and multi-locus sequence typing. A total of 148 cases of meningitis were identified from January 2008 to December 2012, 77 (52 %) of which were due to non-PCV10 isolates, with 50 (52.1 %) from pre-vaccine and 27 (52 %) from post-vaccine periods. In the post-vaccine period, the non-PCV10 serotypes 12F (n=6; 22.2 %), 10A (n=3; 11.1 %), 15B (n=2; 7.4 %) and 18B (n=2; 7.4 %) were the most prevalent. Forty-three isolates (55.8 %) were non-susceptible to one or more antibiotics. Non-susceptibility to penicillin was observed among serotypes 19A (three isolates), 9N (one isolate) and 12F (one isolate). PFGE and multi-locus sequence typing results demonstrated a wide genetic diversity among the isolates. During the early period following PCV10 introduction, no obvious emergence of a particular serotype was evident among non-PCV10 strains. This study underscores the importance of monitoring any changes among non-PCV10 cases after the introduction of PCV10.

This study aimed to examine occurrence and antimicrobial resistance characteristics of Salmonella from pigs, pork and humans in Thailand and Laos provinces. The samples were collected from pigs, carcasses and workers in slaughterhouses, retail pork and butchers in fresh markets and patients in hospitals in Thailand (n=729) and Laos (n=458). A total of 295 of 729 samples (34.6 %) collected in Thailand and 253 of 458 (47.4 %) samples collected in Laos were positive for Salmonella. A total of 548 Salmonella isolates from Thailand (n=295) and Laos (n=253) were further analysed. Serovar Typhimurium was the most common serotype in Thai (34 %) and Laos (20.6 %) samples. Approximately 2.4 % of Thai isolates produced extended-spectrum β-lactamase (ESBL). All the ESBL producers possessed bla CTX-M-14, some of which were horizontally transferred. Class 1 integrons were common in Thai (31.9 %) and Laos (39.1 %) isolates, but none were associated with SGI1. The resistance cassette dfrA12-aadA2 was the most common, while the least common was aadA2-linG (n=1). The dfrA12-aadA2 gene cassette in five isolates and aadA2-linG were located on conjugative plasmid. Three pork isolates were fluoroquinolone resistant and carried an amino acid substitute, Ser-83-Tyr, in GyrA. The qnrS gene was found in 7.1 and 5.5 % of the Thai and Laos isolates, respectively, while qnrB was carried in another Laos isolate (1.9 %). All ESBL producers carried qnrS. In conclusion, multidrug-resistant Salmonella was common in pigs, pork and human samples in this region. The bacteria carried mobile genetic elements and resistance genes on conjugative plasmids that could be readily transferred to other bacterial species.