Bacterial and Fungal Pathogens: New Weapons to Fight Them.

In high-income countries, degenerative diseases are the primary cause of death [...].

Bacterial Sub-Species Typing Using Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry: What Is Promising?

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is routinely used for bacterial identification. It would be highly beneficial to also be able to use the technology as a fast way to detect clinically relevant clones of bacterial species. However, studies to this aim have often had limited success. The methods used for data acquisition, processing and data interpretation are highly diverse amongst studies on MALDI-TOF MS sub-species typing. In addition to this, feasibility may depend on the bacterial species and strains investigated, making it difficult to determine what methods may or may not work. In our paper, we have reviewed recent research on MALDI-TOF MS typing of bacterial strains. Although we found a lot of variation amongst the methods used, there were approaches shared by multiple research groups. Multiple spectra of the same isolate were often combined before further analysis for strain distinction. Many groups used a protein extraction step to increase resolution in their MALDI-TOF MS results. Peaks at a high mass range were often excluded for data interpretation. Three groups have found ways to determine feasibility of MALDI-TOF MS typing for their set of strains at an early stage of their project.

Bacterial identification using a SCIEX 5800 TOF/TOF MALDI research instrument and an external database.

In our current study we were identifying 26 bacterial isolates using a SCIEX 5800 TOF/TOF MALDI instrument and an external database. The results were compared with the results of a Vitek® MS system and in case of discrepancies at the species level 16s rRNA sequencing was performed for further verification.

Upper Versus Lower Gastrointestinal Delivery for Transplantation of Fecal Microbiota in Recurrent or Refractory Clostridium difficile Infection: A Collaborative Analysis of Individual Patient Data From 14 Studies.

GOALS: The aim of this study was to compare upper gastrointestinal (UGI) versus lower gastrointestinal (LGI) delivery routes of fecal microbiota transplantation (FMT) for refractory or recurrent/relapsing Clostridium difficile infection (CDI). BACKGROUND: FMT has been proven to be a safe and highly effective therapeutic option for CDI. Delivery, however, could be via the UGI or LGI routes, and it is unclear as to which route provides better clinical outcome. STUDY: A systematic search for studies that reported the use of FMT for CDI treatment was conducted. Individual patient data that included demographic (age and sex) and clinical (route of FMT delivery, CDI outcome after FMT, and follow-up time) information were obtained. Kaplan-Meier cumulative hazard curves and Cox proportional hazard models were used to assess clinical failure after FMT by the route of delivery. RESULTS: Data from 305 patients treated with FMT (208 via LGI route and 97 via UGI route) for CDI were analyzed. At 30 and 90 days, the risk of clinical failure was 5.6% and 17.9% in the UGI group compared with 4.9% and 8.5% in the LGI delivery route group, respectively. A time-varying analysis suggested a 3-fold increase in hazard of clinical failure for UGI delivery (hazard ratio, 3.43; 95% confidence interval, 1.32-8.93) in the period after 30 days. CONCLUSIONS: FMT delivered via the LGI seems to be the most effective route for the prevention of recurrence/relapse of CDI. A randomized controlled trial is necessary to confirm whether FMT delivered via the LGI is indeed superior to that delivered via the UGI route.

Comparison of Clostridium difficile Ribotypes Circulating in Australian Hospitals and Communities.

Clostridium difficile infection (CDI) is becoming less exclusively a health care-associated CDI (HA-CDI). The incidence of community-associated CDI (CA-CDI) has increased over the past few decades. It has been postulated that asymptomatic toxigenic C. difficile (TCD)-colonized patients may play a role in the transfer of C. difficile between the hospital setting and the community. Thus, to investigate the relatedness of C. difficile across the hospital and community settings, we compared the characteristics of symptomatic and asymptomatic host patients and the pathogens from these patients in these two settings over a 3-year period. Two studies were simultaneously conducted; the first study enrolled symptomatic CDI patients from two tertiary care hospitals and the community in two Australian states, while the second study enrolled asymptomatic TCD-colonized patients from the same tertiary care hospitals. A total of 324 patients (96 with HA-CDI, 152 with CA-CDI, and 76 colonized with TCD) were enrolled. The predominant C. difficile ribotypes isolated in the hospital setting corresponded with those isolated in the community, as it was found that for 79% of the C. difficile isolates from hospitals, an isolate with a matching ribotype was isolated in the community, suggesting that transmission between these two settings is occurring. The toxigenic C. difficile strains causing symptomatic infection were similar to those causing asymptomatic infection, and patients exposed to antimicrobials prior to admission were more likely to develop a symptomatic infection (odds ratio, 2.94; 95% confidence interval, 1.20 to 7.14). Our findings suggest that the development of CDI symptoms in a setting without establishment of hospital epidemics with binary toxin-producing C. difficile strains may be driven mainly by host susceptibility and exposure to antimicrobials, rather than by C. difficile strain characteristics.

Detection of carbapenemase activity in Enterobacteriaceae using LC-MS/MS in comparison with the neo-rapid CARB kit using direct visual assessment and colorimetry.

It has been described that the sensitivity of the Carba NP test may be low in the case of OXA-48-like carbapenamases and mass spectrometry based methods as well as a colorimetry based method have been described as alternatives. We evaluated 84 Enterobacteriaceae isolates including 31 OXA-48-like producing isolates and 13 isolates that produced either an imipenemase (IMP; n=8), New Delhi metallo-ß-lactamase (NDM; n=3), or Klebsiella pneumoniae carbapenemase (KPC; n=2), as well as 40 carbapenemase negative Enterobacteriaceae isolates. We used the Neo-Rapid CARB kit, assessing the results with the unaided eye and compared it with a colorimetric approach. Furthermore, we incubated the isolates in growth media with meropenem and measured the remaining meropenem after one and 2h of incubation, respectively, using liquid chromatography tandem mass spectrometry (LC-MS/MS). Whilst all carbapenemase producing isolates with the exception of the OXA-244 producer tested positive for both the Neo-rapid CARB test using the unaided eye or colorimetry, and the 13 isolates producing either IMP, NDM or KPC hydrolysed the meropenem in the media almost completely after 2h of incubation, the 31 OXA-48-like producing isolates exhibited very variable hydrolytic activity when incubated in growth media with meropenem. In our study, the Neo-Rapid CARB test yielded a sensitivity of 98% for both the traditional and the colorimetric approach with a specificity of 95% and 100% respectively. Our results indicate that the Neo-Rapid CARB test may have use for the detection of OXA-48 type carbapenemases and that it may be particularly important to ensure bacterial lysis for the detection of these weaker hydrolysers.

Asymptomatic Clostridium difficile colonization: epidemiology and clinical implications.

BACKGROUND: The epidemiology of Clostridium difficile infection (CDI) has changed over the past decades with the emergence of highly virulent strains. The role of asymptomatic C. difficile colonization as part of the clinical spectrum of CDI is complex because many risk factors are common to both disease and asymptomatic states. In this article, we review the role of asymptomatic C. difficile colonization in the progression to symptomatic CDI, describe the epidemiology of asymptomatic C. difficile colonization, assess the effectiveness of screening and intensive infection control practices for patients at risk of asymptomatic C. difficile colonization, and discuss the implications for clinical practice. METHODS: A narrative review was performed in PubMed for articles published from January 1980 to February 2015 using search terms 'Clostridium difficile' and 'colonization' or 'colonisation' or 'carriage'. RESULTS: There is no clear definition for asymptomatic CDI and the terms carriage and colonization are often used interchangeably. The prevalence of asymptomatic C. difficile colonization varies depending on a number of host, pathogen, and environmental factors; current estimates of asymptomatic colonization may be underestimated as stool culture is not practical in a clinical setting. CONCLUSIONS: Asymptomatic C. difficile colonization presents challenging concepts in the overall picture of this disease and its management. Individuals who are colonized by the organism may acquire protection from progression to disease, however they also have the potential to contribute to transmission in healthcare settings.

Species identification within Acinetobacter calcoaceticus-baumannii complex using MALDI-TOF MS.

Acinetobacter baumannii, one of the more clinically relevant species in the Acinetobacter genus is well known to be multi-drug resistant and associated with bacteremia, urinary tract infection, pneumonia, wound infection and meningitis. However, it cannot be differentiated from closely related species such as Acinetobacter calcoaceticus, Acinetobacter pittii and Acinetobacter nosocomialis by most phenotypic tests and can only be differentiated by specific, time consuming genotypic tests with very limited use in clinical microbiological laboratories. As a result, these species are grouped into the A. calcoaceticus-A. baumannii (Acb) complex. Herein we investigated the mass spectra of 73 Acinetobacter spp., representing ten different species, using an AB SCIEX 5800 MALDI-TOF MS to differentiate members of the Acinetobacter genus, including the species of the Acb complex. RpoB gene sequencing, 16S rRNA sequencing, and gyrB multiplex PCR were also evaluated as orthogonal methods to identify the organisms used in this study. We found that whilst 16S rRNA and rpoB gene sequencing could not differentiate A. pittii or A. calcoaceticus, they can be differentiated using gyrB multiplex PCR and MALDI-TOF MS. All ten Acinetobacter species investigated could be differentiated by their MALDI-TOF mass spectra.

Surveillance for antimicrobial resistance in Australian isolates of Clostridium difficile, 2013-14.

OBJECTIVES: The objective of this study was to determine the activity of fidaxomicin and comparator antimicrobials against Clostridium difficile isolated from patients with C. difficile infection (CDI) in Australian hospitals and in the community. METHODS: One private and one public laboratory from five states in Australia submitted a total of 474 isolates/PCR-positive stool samples during three collection periods in August-September 2013 (n = 175), February-March 2014 (n = 134) and August-September 2014 (n = 165). Isolate identification was confirmed by selective culture for C. difficile and a proportion of isolates from each state were characterized by PCR for toxin genes and PCR ribotyping. MICs of fidaxomicin and eight comparator antimicrobials were determined for all isolates using agar methodology. RESULTS: Site collection yielded 440 isolates of C. difficile and PCR revealed a heterogeneous strain population comprising 37 different PCR ribotypes (RTs), 95% of which were positive for tcdA and tcdB (A+B+). The most common RTs were 014 (29.8%) and 002 (15.9%). Epidemic RT 027 was not identified; however, small numbers of virulent RTs 078 and 244 were found. Resistance to vancomycin, metronidazole and fidaxomicin was not detected and resistance to moxifloxacin was very low (3.4%). Fidaxomicin showed potent in vitro activity against all 440 isolates (MIC50/MIC90 0.03/0.12 mg/L) and was superior to metronidazole (MIC50/MIC90 0.25/0.5 mg/L) and vancomycin (MIC50/MIC90 1/2 mg/L). CONCLUSIONS: These data confirm the potent in vitro activity of fidaxomicin against C. difficile. Moreover, this study provides an important baseline for ongoing long-term surveillance of antimicrobial resistance and prospective tracking of prominent and emerging strain types.

Outbreaks of multidrug-resistant Acinetobacter baumannii strains in a Kenyan teaching hospital.

Acinetobacter baumannii is a serious nosocomial pathogen with a high propensity to cause outbreaks. Whilst outbreaks of A. baumannii have been reported in many regions worldwide, few data are available from East Africa. In this study, 25 A. baumannii isolates derived from a single institution located in Nairobi, Kenya, between September 2010 and September 2011 were examined. Antimicrobial susceptibility testing was performed by the disc diffusion method and the relatedness among the isolates was examined by pulsed-field gel electrophoresis, repetitive sequence-based PCR (rep-PCR) and multilocus sequence typing. The examined isolates clustered into three distinct groups. The most prevalent sequence type (ST) was ST110 (17 isolates), followed by ST92 (5 isolates) and ST109 (3 isolates). All isolates exhibited resistance to cefepime, ceftazidime, ticarcillin/clavulanic acid, cefotaxime/clavulanic acid, piperacillin/tazobactam, cefoxitin, ciprofloxacin, gentamicin, nitrofurantoin, fosfomycin trometamol, trimethoprim/sulfamethoxazole, amikacin, meropenem and imipenem, with the exception of four isolates. Two isolates belonging to ST92 and two isolates belonging to ST109 were susceptible to amikacin; one of these amikacin-susceptible ST109 isolates was also susceptible to meropenem and imipenem. All isolates were positive for OXA 51-like and all carbapenem-resistant isolates were OXA-23 positive.

Surveillance snapshot of Clostridium difficile infection in hospitals across Queensland detects binary toxin producing ribotype UK 244.

In North America and Europe, the binary toxin positive Clostridium difficile strains of the ribotypes 027 and 078 have been associated with death, toxic megacolon and other adverse outcomes. Following an increase in C. difficile infections (CDIs) in Queensland, a prevalence study involving 175 hospitals was undertaken in early 2012, identifying 168 cases of CDI over a 2 month period. Patient demographics and clinical characteristics were recorded, and C. difficile isolates were ribotyped and tested for the presence of binary toxin genes. Most patients (106/168, 63.1%) were aged over 60 years. Overall, 98 (58.3%) developed symptoms after hospitalisation; 89 cases (53.0%) developed symptoms more than 48 hours after admission. Furthermore, 27 of the 62 (67.7%) patients who developed symptoms in the community ad been hospitalised within the last 3 months. Thirteen of the 168 (7.7%) cases identified had severe disease, resulting in admission to the Intensive Care Unit or death within 30 days of the onset of symptoms. The 3 most common ribotypes isolated were UK 002 (22.9%), UK 014 (13.3%) and the binary toxin-positive ribotype UK 244 (8.4%). The only other binary toxin positive ribotype isolated was UK 078 (n = 1). Of concern was the detection of the binary toxin positive ribotype UK 244, which has recently been described in other parts of Australia and New Zealand. No isolates were of the international epidemic clone of ribotype UK 027, although ribotype UK 244 is genetically related to this clone. Further studies are required to track the epidemiology of ribotype UK 244 in Australia and New Zealand.

Camel Streptococcus agalactiae populations are associated with specific disease complexes and acquired the tetracycline resistance gene tetM via a Tn916-like element.

Camels are the most valuable livestock species in the Horn of Africa and play a pivotal role in the nutritional sustainability for millions of people. Their health status is therefore of utmost importance for the people living in this region. Streptococcus agalactiae, a Group B Streptococcus (GBS), is an important camel pathogen. Here we present the first epidemiological study based on genetic and phenotypic data from African camel derived GBS. Ninety-two GBS were characterized using multilocus sequence typing (MLST), capsular polysaccharide typing and in vitro antimicrobial susceptibility testing. We analysed the GBS using Bayesian linkage, phylogenetic and minimum spanning tree analyses and compared them with human GBS from East Africa in order to investigate the level of genetic exchange between GBS populations in the region. Camel GBS sequence types (STs) were distinct from other STs reported so far. We mapped specific STs and capsular types to major disease complexes caused by GBS. Widespread resistance (34%) to tetracycline was associated with acquisition of the tetM gene that is carried on a Tn916-like element, and observed primarily among GBS isolated from mastitis. The presence of tetM within different MLST clades suggests acquisition on multiple occasions. Wound infections and mastitis in camels associated with GBS are widespread and should ideally be treated with antimicrobials other than tetracycline in East Africa.

Challenges for standardization of Clostridium difficile typing methods.

Typing of Clostridium difficile facilitates understanding of the epidemiology of the infection. Some evaluations have shown that certain strain types (for example, ribotype 027) are more virulent than others and are associated with worse clinical outcomes. Although restriction endonuclease analysis (REA) and pulsed-field gel electrophoresis have been widely used in the past, PCR ribotyping is the current method of choice for typing of C. difficile. However, global standardization of ribotyping results is urgently needed. Whole-genome sequencing of C. difficile has the potential to provide even greater epidemiologic information than ribotyping.

Lack of antigenic diversification of major outer membrane proteins during clonal waves of Neisseria meningitidis serogroup A colonization and disease.

In particular in the 'meningitis belt' of sub-Saharan Africa, epidemic meningococcal meningitis is a severe public health problem. In the past decades, serogroup A lineages have been the dominant etiologic agents, but also other serogroups have caused outbreaks. A comprehensive vaccine based on subcapsular outer membrane proteins (OMPs) is not available. Here, we have investigated whether meningococcal populations overcome herd immunity by changing antigenic properties of their OMPs. Meningococcal isolates were collected in the context of longitudinal studies in Ghana between 2002 and 2008 and in Burkina Faso between 2006 and 2007. Serogroup A strains isolated during two clonal waves of colonization and disease showed no diversification in the genes encoding their PorA, PorB, and FetA proteins. However, we detected occasional allelic exchange of opa genes, as well as wide variation in the number of intragenic tandem repeats, showing that phase variation of Opa protein expression is a frequent event. Altogether we observed a remarkable antigenic stability of the PorA, PorB and FetA proteins over years. Our results indicate that while herd immunity may be responsible for the disappearance of meningococcal clones over time, it is not a strong driving force for antigenic diversification of the major OMPs analyzed here.

Characterization of invasive and colonizing isolates of Streptococcus agalactiae in East African adults.

Ninety-five colonizing isolates and 74 invasive isolates of Streptococcus agalactiae from Kenyan adults were characterized by using capsular serotyping and multilocus sequence typing. Twenty-two sequence types clustering into five clonal complexes were found. Data support the view that S. agalactiae isolates belonging to a limited number of clonal complexes are invasive in adults worldwide.

Independent loss of immunogenic proteins in Mycobacterium ulcerans suggests immune evasion.

The highly immunogenic mycobacterial proteins ESAT-6, CFP-10, and HspX represent potential target antigens for the development of subunit vaccines and immunodiagnostic tests. Recently, the complete genome sequence revealed the absence of these coding sequences in Mycobacterium ulcerans, the causative agent of the emerging human disease Buruli ulcer. Genome reduction and the acquisition of a cytopathic and immunosuppressive macrolide toxin plasmid are regarded as crucial for the emergence of this pathogen from its environmental progenitor, Mycobacterium marinum. Earlier, we have shown the evolution of M. ulcerans into two distinct lineages. Here, we show that while the genome of M. marinum M contains two copies of the esxB-esxA gene cluster at different loci (designated MURD4 and MURD152), both copies are deleted from the genome of M. ulcerans strains belonging to the classical lineage. Members of the ancestral lineage instead retained some but disrupted most functional MURD4 or MURD152 copies, either by newly identified genomic insertion-deletion events or by conversions of functional genes to pseudogenes via point mutations. Thus, the esxA (ESAT-6), esxB (CFP-10), and hspX genes are located in hot-spot regions for genomic variation where functional disruption seems to be favored by selection pressure. Our detailed genomic analyses have identified a variety of independent genomic changes that have led to the loss of expression of functional ESAT-6, CFP-10, and HspX proteins. Loss of these immunodominant proteins helps the bacteria bypass the host's immunological response and may represent part of an ongoing adaptation of M. ulcerans to survival in host environments that are screened by immunological defense mechanisms.