Anti-LPS IgA and IgG can inhibit serum killing of Pseudomonas aeruginosa in patients with cystic fibrosis.

Pseudomonas aeruginosa is one of the principal pathogens implicated in respiratory infections of patients with cystic fibrosis (CF) and non-CF bronchiectasis. Previously, we demonstrated that impaired serum-mediated killing of P. aeruginosa was associated with increased severity of respiratory infections in patients with non-CF bronchiectasis. This inhibition was mediated by high titres of O-antigen-specific IgG2 antibodies that cloak the surface of the bacteria, blocking access to the membrane. Infection related symptomatology was ameliorated in patients by using plasmapheresis to remove the offending antibodies. To determine if these inhibitory ‘cloaking antibodies’ were prevalent in patients with CF, we investigated 70 serum samples from patients with P. aeruginosa infection, and five without P. aeruginosa infection. Thirty-two percent of patients had serum that inhibited the ability of healthy control serum to kill P. aeruginosa . Here we demonstrate that this inhibition of killing requires O-antigen expression. Furthermore, we reveal that while IgG alone can inhibit the activity of healthy control serum, O-antigen specific IgA in patient sera can also inhibited serum-killing. We found that antibody affinity, not just titre, was also important in the inhibition of serum-mediated killing. These studies provide novel insight into cloaking antibodies in human infection and may provide further options in CF and other diseases for treatment of recalcitrant P. aeruginosa infections.

β-lactam resistance associated with β-lactamase production and porin alteration in clinical isolates of E. coli and K. pneumoniae

β-lactam resistance represents a worldwide problem and a serious challenge for antimicrobial treatment. Hence this research was conducted to recognize several mechanisms mediating β-lactam resistance in E. coli and K. pneumoniae clinical isolates collected from Mansoura University hospitals, Egypt. A total of 80 isolates, 45 E. coli and 35 K. pneumoniae isolates, were collected and their antibiotic susceptibility was determined by the Disc diffusion method followed by phenotypic and genotypic detection of extended-spectrum β-lactamases (ESBLs), AmpC β-lactamase, carbapenemase enzymes. The outer membrane protein porins of all isolates were analyzed and their genes were examined using gene amplification and sequencing. Also, the resistance to complement-mediated serum killing was estimated. A significant percentage of isolates (93.8%) were multidrug resistance and showed an elevated resistance to β-lactam antibiotics. The presence of either ESBL or AmpC enzymes was high among isolates (83.75%). Also, 60% of the isolated strains were carbapenemase producers. The most frequently detected gene of ESBL among all tested isolates was blaCTX-M-15 (86.3%) followed by blaTEM-1 (81.3%) and blaSHV-1 (35%) while the Amp-C gene was present in 83.75%. For carbapenemase-producing isolates, blaNDM1 was the most common (60%) followed by blaVIM-1 (35%) and blaOXA-48 (13.8%). Besides, 73.3% and 40% of E. coli and K. pneumoniae isolates respectively were serum resistant. Outer membrane protein analysis showed that 93.3% of E. coli and 95.7% of K. pneumoniae isolates lost their porins or showed modified porins. Furthermore, sequence analysis of tested porin genes in some isolates revealed the presence of frameshift mutations that produced truncated proteins of smaller size. β-lactam resistance in K. pneumoniae and E. coli isolates in our hospitals is due to a combination of β-lactamase activity and porin loss/alteration. Hence more restrictions should be applied on β-lactams usage to decrease the emergence of resistant strains.

Virulence Characteristics and Molecular Epidemiology of Pyogenic Liver Abscess Causing Multidrug Resistant Klebsiella pneumoniae in Wenzhou, China

Background To date, little is known about the virulence characteristics of pyogenic liver abscess (PLA) that cause multidrug resistant (MDR) Klebsiella pneumoniae (K. pneumoniae), which might be due to the rarity of these strains. This study aimed to analyze the virulence characteristics and molecular epidemiology of 12 MDR strains obtained from 163 PLA cases in a tertiary teaching hospital from the perspective of clinical characteristics, virulence phenotypes, and genotypes. Methods The MDR strains were obtained from sterile fluid samples collected from patients with PLA. The antimicrobial susceptibility testing was confirmed by the agar dilution method and microdilution broth method. The virulence phenotypes were analyzed by the growth curves, string test, capsular quantification, serum killing test, biofilm formation assay and infection model. Polymerase chain reaction (PCR) was used to investigate the virulence genotypes. The molecular epidemiology was identified by multilocus sequence typing (MLST). Results The results of growth curves, string test, capsular quantification, serum killing test, biofilm formation assay, and infection model revealed that the virulence phenotypes of the 12 PLA-causing MDR K. pneumoniae were similar to or more obvious than those of the typical hypervirulent K. pneumoniae strains. These MDR strains were mainly non-K1/K2 serotypes and carried multiple virulence genes. The results of MLST illustrated that the MDR strains were categorized into 9 sequence types. Conclusions This is the first study to analyze the virulence characteristics in PLA-causing MDR strains. The data revealed the coexistence of hypervirulence and MDR in PLA-causing MDR K. pneumoniae strains, and the clones of these strains were diverse and scattered. Also, one ST11 carbapenem-resistant hypervirulent strain was identified in PLA.

Negative correlation between virulence and multidrug resistance in intrahospital and community acquired infections by Proteus mirabilis, in Eastern Venezuela

This is the first report for Venezuela of virulence/pathogenicity and resistance factors in intrahospital (HCAI) and community-acquired infections (CAI) by P. mirabilis in two main hospitals from Eastern Venezuela. Virulence factors such as motility, biofilms, and resistance to serum killing (RSK) were determined. Antimicrobial susceptibility allowed classifying the isolates into resistant, multidrug resistant (MDR) and extensively drug-resistant (XDR). P. mirabilis was identified in HCAI in both hospitals mostly from secretions, while some CAI were identified from urine and secretions. Twitching, swarming, biofilm and RSK were identified in many isolates. Eleven antimicrobials showed resistance frequencies from 22-54% in one or both hospitals. A high frequency of MDR isolates was found in these hospitals (60.6 to 56.5%). Strains carrying both blaCTX-M and blaTEM genes were found in one hospital in a frequency of 27.0%. We also found that the frequency of MDR was lower in strains with three or more virulence factors compared to those with fewer factors. Bacteria with swarming showed 5.85 times lower probability of being MDR, and those with twitching, 7.52 times lower probability. Infections by MDR/XDR P. mirabilis strains in HCAI and CAI represent a public health problem that requires effective control and prevention measures to reduce their potential spread and persistence in the population.

A Protective and Pathogenic Role for Complement During Acute Toxoplasma gondii Infection

The infection competence of the protozoan pathogen Toxoplasma gondii is critically dependent on the parasite’s ability to inactivate the host complement system. Toxoplasma actively resists complement-mediated killing in non-immune serum by recruiting host-derived complement regulatory proteins C4BP and Factor H (FH) to the parasite surface to inactivate surface-bound C3 and limit formation of the C5b-9 membrane attack complex (MAC). While decreased complement activation on the parasite surface certainly protects Toxoplasma from immediate lysis, the biological effector functions of C3 split products C3b and C3a are maintained, which includes opsonization of the parasite for phagocytosis and potent immunomodulatory effects that promote pro-inflammatory responses and alters mucosal defenses during infection, respectively. In this review, we discuss how complement regulation by Toxoplasma controls parasite burden systemically but drives exacerbated immune responses locally in the gut of genetically susceptible C57BL/6J mice. In effect, Toxoplasma has evolved to strike a balance with the complement system, by inactivating complement to protect the parasite from immediate serum killing, it generates sufficient C3 catabolites that signal through their cognate receptors to stimulate protective immunity. This regulation ultimately controls tachyzoite proliferation and promotes host survival, parasite persistence, and transmissibility to new hosts.

Virulence Characteristics and Molecular Epidemiology of Pyogenic Liver Abscess Causing Multidrug Resistant Klebsiella pneumoniae in Wenzhou, China

Background: To date, little is known about the virulence characteristics of pyogenic liver abscess (PLA) that cause multidrug resistant (MDR) Klebsiella pneumoniae (K. pneumoniae), and this might be due to that these strains are rare. This study aimed to analyze the virulence characteristics and molecular epidemiology of 12 MDR strains obtained from 163 PLA cases in a tertiary teaching hospital from the perspective of clinical characteristics, virulence phenotypes, and genotypes.Results: The results of growth curves, string test, capsular quantification, serum killing test, biofilm formation assay, and infection model revealed that the virulence phenotypes of the 12 PLA-causing MDR K. pneumoniae were similar or even more obvious than those of typical hypervirulent K. pneumoniae strains. These MDR strains were mainly non-K1/K2 serotypes and carried multiple virulence genes. The results of multilocus sequence typing (MLST) illustrated that the MDR strains were categorized into 9 sequence types.Conclusions: This study is the first to analyze the virulence characteristics in PLA-causing MDR strains. Our data exhibited the coexistence of hypervirulence and MDR in PLA-causing MDR K. pneumoniae strains, and the clones of those PLA-causing MDR strains were diverse and scattered. This study first found one ST11 carbapenem-resistant hypervirulent strain in PLA.

Novel inhibitors of E. coli lipoprotein diacylglyceryl transferase are insensitive to resistance caused by lpp deletion

Lipoprotein diacylglyceryl transferase (Lgt) catalyzes the first step in the biogenesis of Gram-negative bacterial lipoproteins which play crucial roles in bacterial growth and pathogenesis. We demonstrate that Lgt depletion in a clinical uropathogenic Escherichia coli strain leads to permeabilization of the outer membrane and increased sensitivity to serum killing and antibiotics. Importantly, we identify the first ever described Lgt inhibitors that potently inhibit Lgt biochemical activity in vitro and are bactericidal against wild-type Acinetobacter baumannii and E. coli strains. Unlike inhibition of other steps in lipoprotein biosynthesis, deletion of the major outer membrane lipoprotein, lpp, is not sufficient to rescue growth after Lgt depletion or provide resistance to Lgt inhibitors. Our data validate Lgt as a novel druggable antibacterial target and suggest that inhibition of Lgt may not be sensitive to one of the most common resistance mechanisms that invalidate inhibitors of downstream steps of bacterial lipoprotein biosynthesis and transport.

A Tailspike with Exopolysaccharide Depolymerase Activity from a New Providencia stuartii Phage Makes Multidrug-Resistant Bacteria Susceptible to Serum-Mediated Killing

ABSTRACT Providencia stuartii is emerging as a significant drug-resistant nosocomial pathogen, which encourages the search for alternative therapies. Here, we have isolated Providencia stuartii phage Stuart, a novel podovirus infecting multidrug-resistant hospital isolates of this bacterium. Phage Stuart is a proposed member of a new Autographivirinae subfamily genus, with a 41,218-bp genome, direct 345-bp repeats at virion DNA ends, and limited sequence similarity of proteins to proteins in databases. Twelve out of the 52 predicted Stuart proteins are virion components. We found one to be a tailspike with depolymerase activity. The tailspike could form a highly thermostable oligomeric β-structure migrating close to the expected trimer in a nondenaturing gel. It appeared to be essential for the infection of three out of four P. stuartii hosts infected by phage Stuart. Moreover, it degraded the exopolysaccharide of relevant phage Stuart hosts, making the bacteria susceptible to serum killing. Prolonged exposure of a sensitive host to the tailspike did not cause the emergence of bacteria resistant to the phage or to serum killing, opposite to the prolonged exposure to the phage. This indicates that phage tail-associated depolymerases are attractive antivirulence agents that could complement the immune system in the fight with P. stuartii. IMPORTANCE The pace at which multidrug-resistant strains emerge has been alarming. P. stuartii is an infrequent but relevant drug-resistant nosocomial pathogen causing local to systemic life-threatening infections. We propose an alternative approach to fight this bacterium based on the properties of phage tailspikes with depolymerase activity that degrade the surface bacterial polymers, making the bacteria susceptible to the immune system. Unlike antibiotics, phage tailspikes have narrow and specific substrate spectra, and by acting as antivirulent but not bactericidal agents they do not cause the selection of resistant bacteria.

Phenotype, Virulence and Immunogenicity of Edwardsiella piscicida Cyclic AMP Receptor Protein (Crp) Mutants in Catfish Host

Edwardsiella piscicida, a facultative aerobic pathogen belonging to the Enterobacteriaceae family, is the etiological agent of edwardsiellosis that causes significant economic loses in the aquaculture industry. cAMP receptor protein (CRP) is one of the most important transcriptional regulators, which can regulate large quantities of operons in different bacteria. Here we characterize the crp gene and report the effect of a crp deletion in E. piscicida. The crp-deficient mutant lost the capacity to utilize maltose, and showed significantly reduced motility due to the lack of flagella synthesis. We further constructed a ΔPcrp mutant to support that the phenotype above was caused by the crp deletion. Evidence obtained in fish serum killing assay and competitive infection assay strongly indicated that the inactivation of crp impaired the ability of E. piscicida to evade host immune clearance. More importantly, the virulence of the crp mutant was attenuated in both zebrafish and channel catfish, with reductions in mortality rates. In the end, we found that crp mutant could confer immune protection against E. piscicida infection to zebrafish and channel catfish, indicating its potential as a live attenuated vaccine.