Incidence of multidrug resistance and extended-spectrum beta-lactamase expression in community-acquired urinary tract infection among different age groups of patients.

OBJECTIVES: Urinary tract infection (UTI) is a frequent disorder and depends on age and gender. Ineffective empiric treatment of UTI is common when associated with extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae. The aim of the study was to investigate the prevalence of Gram-negative uropathogens of E. coli and K. pneumoniae in different age groups along with the identification of ESBL-producing uropathogens and antimicrobial susceptibility profiles. MATERIALS AND METHODS: A total of 247 uropathogens of E. coli and K. pneumoniae were collected over a period of 1 year (January-December 2015) from various diagnostic centers of Karachi city (Pakistan). Antimicrobial susceptibility analysis was performed by disc diffusion method, and identification of ESBL was performed by double disc synergy test. Categorical data of ESBL and non-ESBL uropathogens were analyzed by Pearson's Chi-square test. RESULTS: The study of 247 patients with community-acquired UTI comprised 72% females and 28% males, illustrating an increased prevalence of UTIs among females. It was also revealed that 90% belonged to the age group of 16-30 years whereas 78% related to the age group of 46-60 years. ESBL was found positive in 33.5% (63/188) of E. coli and 15.25% (9/59) in K. pneumonia, with a significant association i.e., (p =0.007). Amikacin, fosfomycin, imipenem, and tazobactam/piperacillin were found to be the effective treatment options. A significant association was found between ESBL-producing uropathogens against ciprofloxacin, enoxacin, and amoxicillin/clavulanic acid resistance (P < 0.05). CONCLUSIONS: It was concluded that for effective treatment of UTIs, appropriate screening of ESBL and culture sensitivity must be employed instead of empiric treatment.

Ascorbic acid augments colony spreading by reducing biofilm formation of methicillin-resistant Staphylococcus aureus.

OBJECTIVES: Staphylococcus aureus is a Gram-positive pathogen, well known for its resistance and versatile lifestyle. Under unfavourable conditions, it adapts biofilm mode of growth. For staphylococcal biofilm formation, production of extracellular polymeric substances (EPS) is a pre-requisite, which is regulated by ica operon-encoded enzymes. This study was designed to know the impact of ascorbic acid on biofilm formation and colony spreading processes of S. aureus and MRSA. MATERIALS AND METHODS: The isolates of methicillin-resistant S. aureus (MRSA) used in present study, were recovered from different food samples. Various selective and differential media were used for identification and confirmation of S. aureus. Agar dilution method was used for determination of oxacillin and ascorbic acid resistance level. MRSA isolates were re-confirmed by E-test and by amplification of mecA gene. Tube methods and Congo-Red agar were used to study biofilm formation processes. Gene expression studies were carried on real-time reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: The results revealed the presence of mecA gene belonging to SCCmecA type IV along with agr type II in the isolates. In vitro studies showed the sub-inhibitory concentration of oxacillin induced biofilm production. However, addition of sub-inhibitory dose of ascorbic acid was found to inhibit EPS production, biofilm formation and augment colony spreading on soft agar plates. The inhibition of biofilm formation and augmentation of colony spreading observed with ascorbic acid alone or in combination with oxacillin. Moreover, gene expression studies showed that ascorbic acid increases agr expression and decreases icaA gene expression. CONCLUSION: The present study concluded that ascorbic acid inhibits biofilm formation, promotes colony spreading and increases agr gene expression in MRSA.

Identification and growth optimization of a Marine Bacillus DK1-SA11 having potential of producing broad spectrum antimicrobial compounds.

Control of harmful bacteria in food, aquaculture, pharmaceuticals, agriculture, hospitals and recreation water pools are of great global concern. Marine bacteria are an enormous source of bio-controlling agents. The aim of this study was to identify and optimize the growth conditions including effect of different biotic and abiotic factors on antimicrobial activity of strain DK1-SA11 isolated from Qingdao Bay of China Yellow Sea. Microscopic characterization, API® 20E and 50 CHB kit base carbohydrates utilization, 16S rDNA and DNA gyrB gene sequencing studies identified the bacterium as Bacillus subtilis subsp. spizizenii DK1-SA11. Antimicrobial spectrum of cell free supernatant (CFS) has shown antimicrobial activities against all test strains including methicillin-resistant Staphylococcus aureus, E. coli O157:H7, Candida albicans, Klebsiella pneumoniae, Listeria monocytogenes, Vibrio parahaemolyticus, E. coli, Pseudomonas fluorescens, Salmonella typhimurium and Vibrio cholerae. Among all the media tested, Marine Broth 2216 was found to be the best medium for bacterial growth and production of antibacterial compounds. The other optimum conditions for growth were pH:7 and incubation temperature: 25°C with < 180 rpm for 60-72 h. Out of 49 different carbohydrates tested, D-mannose increases the antibacterial activity by 33.3% while D-arabitol decreases it by 44.4%. Crude CFS showed activity even after three months of storage below -20°C and boiling for 10 min, whereas it loses 100% of its antimicrobial activity after enzymatic treatments of lipase, trypsin and papain. The production of antimicrobial compounds and broad spectrum of antimicrobial activity against all tested pathogens suggested that the strain DK1-SA11 can be used as a source for probiotics, synbiotics and antibiotics.

Antibacterial fatty acids destabilize hydrophobic and multicellular aggregates of biofilm in S. aureus.

Present study is based on 20 methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from different food items. These isolates were identified on the basis of colony morphology, Gram staining and growth on different selective and differential media. Studies on 16S RNA and positive reactions on DNase agar and Prolex Latex Agglutination system confirm it as Staphylococcus aureus. Oxacillin susceptibility testing and PCR with mecA gene-specific primer results showed that these isolates are MRSA-carrying mecA gene that belongs to SCCmecA type IV and also harbor agr type II. Phenotypic study revealed that these isolates adopt biofilm mode of growth after exposure to subinhibitory doses of oxacillin. The biofilm and cell surface hydrophobicity have a strong correlation. It was noticed that affinity to hexadecane (apolar-solvent) of planktonic cells was low, suggesting its hydrophilic character. However, as the cells are exposed to oxacillin, they adopt biofilm mode of life and the affinity to apolar solvent increases, indicating a hydrophobic character. In biofilm consortia, the cells with more hydrophobic surfaces show incomplete septation and produce multicellular aggregates. This is due to reduced expression of atl gene. This was confirmed by real-time PCR studies. Moreover, the planktonic or wild-type phenotypes of these isolates were more tolerant to antibacterial effect of the fatty acids used; that is, cis-2-decanoic acid and cis-9-octadectanoic acid. These fatty acids were more effective against biofilms. After exposure to these fatty acids, established biofilms were dispersed and surviving cells were unable to readopt biofilm mode of life. The planktonic or wild-type phenotypes produce fatty acid-modifying enzyme (FAME) to inactivate the bactericidal activity of fatty acids by esterification to cholesterol. The biofilm indwellers are metabolically inactive and unable to produce FAME; hence, they are vulnerable to antibiofilm effect of cis-2-decanoic acid and cis-9-octadectanoic acid.

Small colony variants have a major role in stability and persistence of Staphylococcus aureus biofilms.

The present study was conducted to investigate the significance of small colony variants (SCVs) in biofilm life cycle of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA). All of these MRSA and MSSA isolates were recovered from different food commodities. Molecular typing showed that 21 MRSA isolates carry SCCmecA type IV and belong to agr type II. Out of 15 MSSA isolates, 7 were found to carry agr type II, 5 agr type I and 2 agr type III. All of the MRSA isolates studied adopted biofilm mode of growth after exposure to sublethal doses of oxacillin. MSSA isolates, on the other hand, were biofilm producers by nature, that is, without exposure to any stress. The biomass of the biofilm reaches its maximum thickness after 48 h of incubation at 35 °C. It was noticed that biofilm population consists of wild type and SCVs. Moreover, the number of SCVs increases with the age of biofilm. The SCVs of MRSA were unable to readopt biofilm mode of growth independently, irrespective of the presence or absence of oxacillin. The SCVs of MSSA, on the other hand, quickly revert to normal life just after a single subculture and show biofilm formation without any stress. Molecular studies showed a parallel reduction in the expression of the genes icaA, sigß and sarA, and also in the extracellular matrix production in SCVs of MRSA. This might be due to oxacillin as it seems to be a stress factor responsible for induction of biofilm formation in MRSA isolates. Contrary to the wild type, SCVs are metabolically inactive and do not respond to oxacillin, which is only active against the growing cells. Therefore, stress-responsive genes, that is, sigß and sarA, are not induced. Conversely, MSSA isolates are natural biofilm producers without induction through any known factors.

Biofilm formation and dispersal of Staphylococcus aureus under the influence of oxacillin.

A total of 180 food borne isolates of methicillin resistant Staphylococcus aureus (MRSA) (oxacillin MICs 864 µg/ml) were used in the present study to investigate the effect of oxacillin on biofilm formation and its detachment process. Majority (98.3%) of these isolates were found to carry icaA gene. Out of 180 isolates 35.5% were identified as MRSA and 64.4% were methicillin sensitive S. aureus (MSSA). Biofilm studies by con-red agar and tube methods revealed that 57% of the MRSA isolates were biofilm producers. Polymerase chain reaction studies suggested that all of the biofilm positive MRSA isolates belong to SCCmec type IV and carry agr type II. This showed the strong association of SCCmec IV agr type II and biofilm formation in food borne MRSA. Conversely, only 13.7% of the MSSA isolates were biofilm positive and majority was found to carry agr type II. It has been noticed that oxacillin has regulatory effect on icaA expression and induce the icaA dependent polysaccharide intracellular adhesin (PIA) production and biofilm formation. This was confirmed by Real Time PCR studies of MRSA and MSSA isolates. Quantitative analysis showed that most of the MRSA isolates started biofilm formation after 24 h of incubation in the presence of sub-inhibitory concentration of oxacillin and achieved highest adhesion on glass slide after 48 h. The control in the absence of oxacillin showed slow conversion from planktonic to biofilm mode of growth (Table 1). Another novel feature of most of these biofilm producing isolates is the reduction in (Optical Density) OD, which is noticed after 48 h of incubation. Possibly, after 48 h oxacillin loses its toxicity or consumed the cells re-adapt to the planktonic state, possibly, by the activation of accessory gene regulator A (agrA) which has an important role in biofilm dispersal.

Effect of stress on biofilm formation by icaA positive and negative strains of methicillin resistant Staphylococcus aureus.

OBJECTIVE: To determine the effect of physical and chemical stress factors e.g. antibiotics, NaCl, glucose, heat shock, cold shock and sonic waves on biofilm formation by icaA positive and negative strains of Methicillin resistant Staphylococcus (S.) aureus. STUDY DESIGN: Experimental study. PLACE AND DURATION OF STUDY: Microbiological Analytical Centre, Pakistan Council of Scientific and Industrial Research (PCSIR) Laboratories Complex, Karachi, from January to December 2010. METHODOLOGY: One strain of Staphylococcus aureus labelled as FA was isolated from a food sample and the other strain labelled as CL was a clinical strain. Biofilm assays were performed in brain-heart infusion (BHI) medium and in BHI supplemented with 7% NaCl, 5% glucose, or sub-inhibitory concentrations of Vancomycin, Oxacillin, Ampicillin, Tetracycline, Erythromycin, Rifampicin and Ciprofloxacin. Polymerase chain reaction (PCR) was used for screening of the icaA and mecA genes. RESULTS: The FA and CL were identified as MRSA carrying mecA gene. The strain FA showed biofilm formation without any treatment and was found to carry icaA gene contrary to CL, that does not contain this gene therefore, is unable to produce biofilm under normal conditions without any stress. The use of sub-lethal doses of cell wall active antibiotics, exposure to 7% NaCl, sonication, and heat shock were found to augment biofilm quantity in FA, an icaA positive strain and induce biofilm mode of growth in CL, an icaA negative strain. Anti-protein synthesis antibiotics did not show any effect on biofilm formation process in icaA positive or negative strains. CONCLUSION: There is a role of anti-cell wall factors i.e. sonication, heat shock, NaCl and antibiotics in the induction of biofilm mode of growth in MRSA and Methicillin sensitive S. aureus. The factors which partially damage bacterial cell wall, equally, induce biofilm formation in icaA positive or negative S. aureus.