The RND Efflux Pump Gene Expression in the Biofilm Formation of Acinetobacter baumannii.

Multidrug resistant (MDR) Acinetobacter baumannii is a critical opportunistic pathogen in healthcare-associated infections (HAI). This is attributed to several factors, including its ability to develop biofilms that can enhance antimicrobial resistance (AMR) in addition to creating an environment for horizontal transfer of antibiotic resistance genes. The role of the efflux pump in biofilm formation is important for studies on alternative treatments for biofilms. One of the significant efflux pump families is the RND efflux pump family, which is common in Gram negative bacteria. The aim is to study the role of the RND efflux pump in biofilm formation by A. baumannii. The biofilm formation potential of thirty-four MDR A. baumannii isolates was evaluated by crystal violet assays. The effect of efflux pump inhibition and activation was studied using the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and the RND efflux pump substrate levofloxacin (at sub-MIC), respectively. The isolates were genotypically grouped by enterobacterial repetitive intergenic consensus (ERIC) typing and the expression of adeABC, adeFGH, and adeIJK efflux pump genes was measured by qPCR. Overall, 88.2% (30/34) of isolates were biofilm producers (the phenotype was variable including strong and weak producers). Efflux pump inhibition by CCCP reduced the biofilm formation significantly (p < 0.05) in 17.6% (6/34) of some isolates, whereas sub-MICs of the substrate levofloxacin increased biofilm formation in 20.5% (7/34) of other isolates. Overexpression of the three RND efflux pump genes was detected in five out of eleven selected isolates for qPCR with remarkable overexpression in the adeJ gene. No correlation was detected between the biofilm phenotype pattern and the RND efflux pump gene expression in biofilm cells relative to planktonic cells. In conclusion, the role of the RND efflux pumps AdeABC, AdeFGH, and AdeIJK in biofilm formation does not appear to be pivotal and the expression differs according to the genetic background of each strain. Thus, these pumps may not be a promising target for biofilm inhibition.

Prevalence of extended-spectrum ß-lactamases, AmpC, and carbapenemases in Proteus mirabilis clinical isolates.

BACKGROUND: Proteus mirabilis is an opportunistic pathogen, causing a variety of community-acquired and nosocomial illnesses. It poses a potential threat to patients via the production of ß-lactamases, which decrease the efficacy of antimicrobial treatment and impair the management of its pathogenicity. Hence, this study was established to determine the prevalence of extended-spectrum ß-lactamases (ESBLs), AmpC, and carbapenemases of P. mirabilis isolated from various clinical specimens. RESULTS: Proteus mirabilis was identified in 20.7% (58/280) of specimens. ESBL producers were present at a rate of 51.7% (30/58). All AmpC-positive isolates (n = 20) produced ESBLs as well, so 66.7% of ESBL-producing isolates coproduced AmpC enzymes. The modified Hodge test confirmed carbapenemase production in six out of seven imipenem nonsusceptible isolates. Of these, only two (5.7%) isolates were also ESBL-and AmpC-positive. Antibiotic resistance reached the highest level for cotrimoxazole (62.1%, n = 36/58 isolates) and the lowest for imipenem (12.1%, n = 7/58 isolates). The levels of multidrug-resistant (MDR) was 41.4% among the tested isolates. The blaSHV (83.3%), blaAmpC (80%), and blaVIM-1 (50%) were the most detected genes in phenotypically confirmed ESBL-, AmpC-, and carbapenemase-producing isolates, respectively. Besides, more than a half of the tested P. mirabilis strains (53%) coproduced ESBLs and AmpC. Moreover, two isolates coproduced ESBLs and AmpC together with carbapenemases. Furthermore, dendrogram analysis showed great genetic divergence based on the 21 different enterobacterial repetitive intergenic consensus (ERIC) patterns (P1-P21) through the 34 ß-lactamase producers. ERIC analysis distinguished clonal similarities between isolates 21 and 22 in P2 and 9 and 10 in P4, which were isolated from the same clinical source and possessed similar patterns of ß-lactamase-encoding genes. CONCLUSION: Hence, there is an urgent need to monitor hospitalized patients and improve healthcare in order to reduce the incidence of infection and outbreaks of infection with antibiotic-resistant Proteus.

Co-Existence of Carbapenemase-Encoding Genes in Acinetobacter baumannii from Cancer Patients.

INTRODUCTION: Acinetobacter baumannii is an opportunistic pathogen, which can acquire new resistance genes. Infections by carbapenem-resistant A. baumannii (CRAB) in cancer patients cause high mortality. METHODS: CRAB isolates from cancer patients were screened for carbapenemase-encoding genes that belong to Ambler classes (A), (B), and (D), followed by genotypic characterization by enterobacterial-repetitive-Intergenic-consensus-polymerase chain reaction (ERIC-PCR) and multilocus-sequence-typing (MLST). RESULTS: A total of 94.1% of CRAB isolates co-harbored more than one carbapenemase-encoding gene. The genes blaNDM, blaOXA-23-like, and blaKPC showed the highest prevalence, with rates of 23 (67.7%), 19 (55.9%), and 17 (50%), respectively. ERIC-PCR revealed 19 patterns (grouped into 9 clusters). MLST analysis identified different sequence types (STs) (ST-268, ST-195, ST-1114, and ST-1632) that belong to the highly resistant easily spreadable International clone II (IC II). Genotype diversity indicated the dissemination of carbapenem-hydrolyzing, ß-lactamase-encoding genes among genetically unrelated isolates. We observed a high prevalence of metallo-ß-lactamase (MBL)-encoding genes (including the highly-resistant blaNDM gene that is capable of horizontal gene transfer) and of isolates harboring multiple carbapenemase-encoding genes from different classes. CONCLUSION: The findings are alarming and call for measures to prevent and control the spread of MBL-encoding genes among bacteria causing infections in cancer patients and other immunocompromised patient populations.

Antimicrobial and Antibiofilm Activities of Probiotic Lactobacilli on Antibiotic-Resistant Proteus mirabilis.

The emergence of biofilm-forming, multi-drug-resistant (MDR) Proteus mirabilis infections is a serious threat that necessitates non-antibiotic therapies. Antibiotic susceptibility and biofilm-forming activity of P. mirabilis isolates from urine samples were assessed by disc diffusion and crystal violet assays, respectively. Antimicrobial activities of probiotic Lactobacilli were evaluated by agar diffusion. Antibiofilm and anti-adherence activities were evaluated by crystal violet assays. While most P. mirabilis isolates were antibiotic-resistant to varying degrees, isolate P14 was MDR (resistant to ceftazidime, cefotaxime, amoxicillin-clavulanic acid, imipenem, ciprofloxacin, and amikacin) and formed strong biofilms. Cultures and cell-free supernatants of Lactobacillus casei and Lactobacillus reuteri exhibited antimicrobial and antibiofilm activities. The 1/16 concentration of untreated supernatants of L. casei and L. reuteri significantly reduced mature biofilm formation and adherence of P14 by 60% and 72%, respectively (for L. casei), and by 73% each (for L. reuteri). The 1/8 concentration of pH-adjusted supernatants of L. casei and L. reuteri significantly reduced mature biofilm formation and adherence of P14 by 39% and 75%, respectively (for L. casei), and by 73% each (for L. reuteri). Scanning electron microscopy (SEM) confirmed eradication of P14's biofilm by L. casei. L. casei and L. reuteri could be utilized to combat Proteus-associated urinary tract infections.

Probiotic Lactobacillus sp. inhibit growth, biofilm formation and gene expression of caries-inducing Streptococcus mutans.

Streptococcus mutans contributes significantly to dental caries, which arises from homoeostasic imbalance between host and microbiota. We hypothesized that Lactobacillus sp. inhibits growth, biofilm formation and gene expression of Streptococcus mutans. Antibacterial (agar diffusion method) and antibiofilm (crystal violet assay) characteristics of probiotic Lactobacillus sp. against Streptococcus mutans (ATCC 25175) were evaluated. We investigated whether Lactobacillus casei (ATCC 393), Lactobacillus reuteri (ATCC 23272), Lactobacillus plantarum (ATCC 14917) or Lactobacillus salivarius (ATCC 11741) inhibit expression of Streptococcus mutans genes involved in biofilm formation, quorum sensing or stress survival using quantitative real-time polymerase chain reaction (qPCR). Growth changes (OD600) in the presence of pH-neutralized, catalase-treated or trypsin-treated Lactobacillus sp. supernatants were assessed to identify roles of organic acids, peroxides and bacteriocin. Susceptibility testing indicated antibacterial (pH-dependent) and antibiofilm activities of Lactobacillus sp. against Streptococcus mutans. Scanning electron microscopy revealed reduction in microcolony formation and exopolysaccharide structural changes. Of the oral normal flora, L. salivarius exhibited the highest antibiofilm and peroxide-dependent antimicrobial activities. All biofilm-forming cells treated with Lactobacillus sp. supernatants showed reduced expression of genes involved in exopolysaccharide production, acid tolerance and quorum sensing. Thus, Lactobacillus sp. can inhibit tooth decay by limiting growth and virulence properties of Streptococcus mutans.