Evaluation of oxacillin resistant screening agar for rapid conventional screening of methicillin resistant staphylococcus aureus in infection control unit

Rapid assessment of clinical specimens for the presence of methicillin resistant Staphylococcus aureus [MRSA] is an important part of the infection control measures taken to control the spread of MRSA and thus, to decrease hospitalization costs. A novel medium, Oxacillin resistant screening Agar [ORSA] medium, was evaluated for the screening of specimens for MRSA in our infection control unit. Swabs of the nose, throat, perineum, and other infected sites [1527] were inoculated onto the ORSA medium and into an enrichment broth [Muller- Hinton broth supplemented with Na Cl and oxacillin[OX-MH broth]]. After 24h of incubation, the enrichment broth was sub-cultured onto one ORSA plate and one lipovitellin chapman salt agar plate. All MRSA isolates were confirmed by PCR amplifying MecA gene. Out of 1527 specimens, 242 [16%] were positive for MRSA. A sensitivity of [70%] was obtained when ORSA medium was used alone as a primary culture, whereas the sensitivity was 88% when a single selective enrichment was used before subculture on ORSA. Among the 414 blue colonies observed on ORSA plates, only 48% were found to be MRSA, 42% were coagulase - negative staphylococci, 4.4% were Enterococcus species, and 3.3% were methcillin sensitive S .aureus. Among 43 MRSA colonies, 21 [49%] were visible only after 48 h of incubation. However, when ORSA plate was used for subculture, 88% of blue colonies were detected after only 24 h of incubation. In conclusion, the ORSA is suitable for screening of MRSA in a simple laboratory of infection control unit with the advantage of the ease of recognition of mannitol fermenting bacteria. An enrichment broth is still needed to ensure a good sensitivity for the recovery of MRSA, and incubation time of 48 h is required for primary culture on ORSA medium

Prevalence and risk factors of extended-spectrum [beta]-lactamase producing Klebisella pneumoniae in neonatal intensive care unit

Extended-spectrum [beta]-lactamase producing [ESBL] Klebsiella pneumoniae [K pneumoniae] is an important cause of nosocomial infections in neonatal intensive care units [NICUs]. Therefore, the aim of this study was to determine: [i] the prevalence of ESBL-producing K pneumoniae in NICU in Pediatric Department at Zagazig University Hospitals, [ii] the frequency of SHV-1 and SHV-2 genes acquisition among ESBL-producing K pneumoniae isolates, [iii] the risk factors associated with ESBL-producing K pneumoniae infection and [iv] the clinical outcomes of infected infants. This study was conducted over a period of one year on all neonates without evidence of any sepsis on admission. Clinical specimens were collected from blood, urine, sputum, cerebrospinal fluid, swabs from wounds and throat and endotracheal tube aspirates, in addition to environmental samples. Bacterial isolates were identified by colonial morphology, Gram's stain and standard biochemical tests. Antibiotic susceptibility was determined by Kirby-Bauer's disc diffusion method, and phenotypic confirmation of ESBL production was done by the double disc synergy test and E test. Genetic detection of SHV-1and SHV-2 genes in ESBL producing K pneumoniae isolates was done by polymerase chain reaction [PCR] and restriction fragment length polymorphism [RFLP] analyses. Risk factors associated with ESBL-producing K pneumoniae infection were analyzed by both univariate and multivariate logistic regression methods. The incidence of total nosocomial infection was 36.2% [122/337]. K pneumoniae was cultured from 24 [7.1%] of the studied infants; of them, 15 [62.5%] were ESBL producer. By RFLP analysis, PCR amplicons revealed the presence of SHV-2 gene in all 15 isolates [100%], while 6 isolates [40%] revealed the presence of both SHV-1 and SHV-2 genes. Independent risk factors for ESBL-producing K pneumoniae infection were mechanical ventilation [P=0.004], birth weight 15 days [P=0.03], total parenteral nutrition [P=0.03] and previous use of oxyimino-antibiotics [P= 0.04]. ESBL producing K pneumoniae infection was associated with higher mortality [26 verus 10% p= 0.04] and prolonged hospitalization for those who survived [P=0.03]. On the other hand, out of the 343 environmental cultures taken from potential sources of infection, 8 isolates [2.3%] of K pneumoniae were identified; 4 from suction tubes, 2 from the incubators and 2 isolates from the hands of HCWs. Five [62.5%] of these isolates were ESBL producers. From this study, it has been concluded that ESBLproducing K pneumoniae infection is a significant source for mortality and morbidity in infants admitted to NICU, and the use of oxyimino-antibiotics is a significant risk factor for infection. Further studies are needed to explore the clinical significance of the SHV-1 and SHV-2 genes

use of polymerase chain reaction for rapid detection of susceptibility of candida species to fluconazole in positive blood cultures

Disseminated fungal infection is a significant cause of mortality in hospitalized patients. In view of both the delay in obtaining identification by conventional methods following blood-culture positivity in patients with candidaemia and the close relationship between species and fluconazole [FLC] susceptibility, early speciation of positive blood cultures has the potential to influence therapeutic decisions. Therefore, the aim of this work was to evaluate the rapid and sensitive real-time PCR method to distinguish the FLC-sensitive from FLC-resistant species of Candida directly from positive blood-culture bottles. A total of 31 consecutive Candida-positive blood-culture sets were collected, in addition to 8 Candida-negative blood-culture bottles were used as negative control samples. Three TaqMan-based real-time PCR assays were used to identify up to six Candida species directly from Bact/Alert blood-culture bottles that showed yeast cells on Gram staining at the time of initial positivity and further identified by standard phenotypic methods. Target sequences in the rRNA gene complex were amplified, using a consensus two-step PCR protocol, to identify Candida albicans, Candida parapsilosis, Candida tropicalis, Candida dubliniensis, Candida glabrata and Candida krusei. The first four of these [FLC-sensitive group] were identified in a single reaction tube using one fluorescent TaqMan probe targeting 18S rRNA sequences conserved in the flour species. The FLC-resistant species [C. krusei and C.glabrata] were detected in two further reactions, each with species specific probe. This method was performed on 31 Candida-positive and 8 Candida-negative blood-culture bottles and the results were 100% concordant with those of phenotypic identification carried out concomitantly. In conclusion, the real-time PCR assay significantly reduces the time required to identify the presence and type of Candida species in blood cultures from more than 72 hours for conventional phenotypic methods to less than 3 hours, and consequently allows optimization of the antifungal regimen at an earlier stage

Evaluation of fluorescence in situ hybridization for rapid identification of microorganisms in positive blood cultures

Rapid identification of microorganisms in blood cultures is necessary to optimize suitable treatment at an early stage. Fluorescence in situ hybridization [FISH] can reduce the time to identification of pathogens in growth-positive blood cultures. So this work aimed to: [i] evaluate the use of FISH with oligonucleotide probes for identification of pathogens in growth -positive blood cultures, [ii] compare the time taken by FISH and traditional methods for identification of pathogens in growth -positive blood cultures, and [iii] determine the potential clinical benefits of FISH technique. In this study, 120 blood cultures with a positive growth index as determined by the automated blood culture system were examined simultaneously by both traditional laboratory methods and FISH method. For this purpose, oligonucleotide probes that are complementary to portions of the 16S or 23S rRNA of the specific bacteria and of 18S or 26S rRNA of the specific yeasts were used, and allowed identification of about 95% of pathogens known to be associated with bacteremia. For all 120 blood cultures, microorganisms were grown after 1 day and identification to the family, genus, or species level was achieved after 1-3 days, while 108 samples [90%] were similarly identified by FISH within 3-4 h only. Staphylococci were identified in 67 of 69 samples [97%], Streptococci and Enterococci in 11 of 11 samples [100%], gram - negative rods in 25 of 34 samples [74%] and fungi in 4 of 4 samples [100%]. The sensitivity and specificity of individual probes exceeded 95% , except for the Enterobacteriaceae probe [93%]. Cross hybridization occurred with the Klebsiella pneumoniae probe for Klebsiella oxytocic. The time gain of FISH compared to that of culture identification was more than 18 h for bacteria and 42 h for yeasts. In conclusion, FISH is a rapid and reliable method for identification of majority of pathogens in blood cultures from septicemic patients. With further extension of the number of probes and a reduction in the turnaround time, FISH will become a very useful diagnostic tool in the diagnosis of bloodstream infections

Phenotyping and genotyping of pseudomonas aeruginosa urine isolates in Zagazig University Hospitals

Pseudomonas aeruginosa [P. aeruginosa] is an important nosocomial pathogen. therefore sensitive typing techniques are required for its control. Seventy six isolates of P.aeruginosa were recovered from midstream urine specimens obtained from the different Departments in the Zagazig University Hospitals [ZUHs]. Five isolates were polymorphic while the remaining 71 isolates were unimorphic. Different morphotypes of the same isolates were studied separately. In addition, 10 environmental isolates were obtained from the wards of the Urology Department. All isolates and their morphological variants were characterized by morphotyping on primary isolation, biochemical reactions using API 20 NE system, serotyping, antimicrobial susceptibility typing and genotyping using enterobacterial repetitive intergenic consensuspolymerase chain ration [ERIC-PCR] and random amplification of polymorphic DNA [ RAPD] using API2H primer. The unimorphic clinical and environmental isolates belonged to morphotype 1 [56.3% and 60%, respectively], morphotype 2 [22.5 % and 30%, respectively] and morphotype 4 [19.7% and 10%. respectively]. For the clinical and environmental isolates, including morphological variants, 14.1% and 30%, respectively showed atypical biochemical reactions. All were esculin positive. Forty seven percent of the clinical isolates and variants were serologically typable and none of the environmental isolates was typable. Among the typable clinical isolates and variants, serotypes 4 [17.1%] and 6 [7.9%] were the commonest. All isolates could be grouped under 14 antimicrobial susceptibility types. ERIC-PCR yielded 31 typing patterns. Twenty one patterns comprised the clinical isolates and their morphological variants, and 10 patterns comprised the environmental isolates. RAPD by API2H primer yielded 13 types for both clinical and environmental isolates. We concluded that ERIC-PCR was the most discriminatory of all the phenotypic and molecular methods used in this study for typing P.aeruginosa. On the other hand, RAPD using API2H primer seems to be useful for further subtype isolates of the same ERIC pattern. Furthermore. P.aeruginosa isolates of the same genotype can be discriminated by using phenotypic methods like serotyping and antimicrobial susceptibility typing. Interestingly, the majority of morphological variants obtained on primary culture from the same midstream urine samples may represent different strains rather than phenotypic plasticity of the same strain. Therefore, antibiograms should be performed separately for such colonial variants. Moreover, in our study, the environmental isolates of P. aeruginosa played a little role, if any, in the spread of P. aeruginosa nosocomial urinary tract infection