Oxacillin Disk Diffusion Test Research Articles

Methicillin, β-lactams, and Clindamycin Resistance Profiles of Staphylococcus aureus Strains Isolated from Patients with UTI in Bechar Province (Algeria)

Introduction: Staphylococcus aureus is a bacterial pathogen responsible for many dis-eases. Multidrug resistance in S aureus has become common worldwide and is a serious clinical issue in the treatment and care of S. aureus-infected patients. S. aureus uropathogenic strains isolated from patients with UTI in Bechar province (Algeria) were phenotypically identified and evaluated for multiple drug resistance (MDR) patterns against recommended antimicrobial drugs. Methods: Through selective media, the uropathogenic S. aureus strains were isolated by analyzing a total of 211 urine samples, then subjected to various biochemical identification tests. Multidrug resistance pattern (MRSA, MLSB, and β-lactamase production) among Staphylococcus aureus uropathogenic strains was performed using phenotypic tests. : A total of 50 S. aureus strains were isolated, i.e., 40,48% of all the isolated uropathogenic agents estimated at 126 isolates. More than 69% of the women had a urinary tract infection, i.e., a sex ratio (SR) of 0,43. The susceptibility test results showed that the isolated S. aureus strains were resistant to various antibiotics of the β-lactams class (penicillin, cefoxitin, and oxacillin) and were susceptible to gentamicin, chloramphenicol, co-trimoxazole, vancomycin, ofloxacin, and ciprofloxacin. Cefoxitin disk diffusion and agar screening tests showed that all the isolated S. aureus strains were qualified as MRSA at the rate of 100% (50/50), whereas a rate of 74% (37/50) was granted to MRSA using oxacillin disk diffusion test. The highest percentage of MDR-MRSA was observed in class three of antibiotics 12 (24%). The lowest percentage was observed in classes 8 and 9 of antibiotics 1 (2%). For MDR-MRSA, the MAR index ranged from 0,27 to 1,0. Results:: Penicillin disk diffusion (DD) and DDST (PI/PIT) tests showed a production rate of β-lactamase in the range of 94% and 80%, respectively. 85,1% (40/50) of penicillin-resistant strains were positive for DDST. Thus, the D-test among MRSA strains shows a rate of 38% (19/50) of cMLSB and no strain attributed to the iMLSB phenotype. Conclusion: This is an alarming rate, confirming the concern that is still being discussed within the medical teams of the national health sector as well as the Algerian antimicrobial resistance network, which requires prompt intervention to update a new strategy to fight antimicrobial re-sistance.

On-farm epidemiology, virulence profiling, and molecular characterization of methicillin-resistant Staphylococcus aureus at goat farms

Antimicrobial resistance (AMR) becomes a challenging issue that limits the therapeutic options for both veterinary and public health professionals. The current study aimed to investigate the on-farm epidemiology, antibiotics resisting profiling, virulence analysis, and molecular detection of methicillin-resistant Staphylococcus aureus (MRSA) at the caprine-human interface. A total of 768 goat milk samples and 94 skin swabs from farm personnel were collected from 30 goat flocks and processed for isolation of S. aureus. The study isolates were confirmed as MRSA based on the oxacillin and cefoxitin disc diffusion test and the presence of mecA gene. MRSA isolates of goats and human origin were characterized and further evaluated for the presence of virulence genes responsible for intramammary infections and public health hazards. The results revealed 26.82 % and 27.79 % goat milk samples and human samples positive for S. aureus, respectively. A higher MRSA prevalence of 35.92 % and 10.71 % was found in goat and human isolates respectively. The phylogenetic analysis revealed a lesser extent of homology in mecA gene of S. aureus isolates at the caprine-human interface. Moreover, this study revealed strong evolutionary connection between the study isolates and MRSA isolates of Pakistani cattle and buffalo while the in-silico protein analysis showed that all sequences have the same protein motifs resembling penicillin binding protein 2a. The risk factors analysis revealed that teat length, drainage system, hygienic measures during milking, use of teat dip, teat injury, and veterinary services were significantly associated with subclinical mastitis in goats. A total of 43.24 % of local MRSA isolates showed multi-drug resistance (MDR). The isolates showed higher resistance to oxytetracycline followed by gentamicin and vancomycin while moxifloxacin, and linezolid were among the susceptible antibiotics. Local MRSA isolates carried virulence markers (nuc and coag genes) and biofilm-associated icaA (43.24 %) and icaD (29.73 %) genes which are responsible for the intramammary infection. The local isolates also carried the virulence genes of public health concern including the enterotoxin C (sec) gene (24.3 %), enterotoxins B (seb) gene (5.41 %), and enterotoxin D (sed) gene (2.7 %). Enterotoxins A (sea) and E (see) genes were not detected in any isolate. The study concluded that MRSA is an emerging and prevailing pathogen in dairy goats with a high potential to transmit to associated human beings. The presence of a variety of virulence factors as well as the associated antibiotic resistance makes MRSA a potential threat at animal-human interface and thus demands further research.

Comparative Susceptibility of Pathogenic Methicillin-Resistant and Methicillin-Susceptible Staphylococcus pseudintermedius to Empirical Cotrimazole for Canine Pyoderma.

The prevalence of methicillin-resistant Staphylococcus pseudintermedius (MRSP) that causes pyoderma has been gradually shifting, according to many surveillance studies, with annual changes. The empirical cotrimazole regimen remains interesting, but research on cotrimazole susceptibility to MRSP is limited. The objective of this study was to evaluate the susceptibility of cotrimazole to canine pyoderma MRSP isolates. Sixty isolates of S. pseudintermedius were identified as 16 MRSP and 44 methicillin-susceptible S. pseudintermedius (MSSP) using an oxacillin disk diffusion test and VITEK 2 system with VITEK GP card. Using the VITEK 2 system with a VITEK AST-GP81 card, the susceptibility rates of MRSP (15.00%) and MSSP (35.00%) to cotrimazole was observed. The median MIC of cotrimazole on MSSP (median, ≤10; IQR, 10-320) was lower than that of MRSP (median, ≥320; IQR, 10-320) (p = 0.5889, Mann-Whitney test). Percent attainment of PK/PD targets in MRSP (q 12 h, 43.75; q 8 h, 43.75) were lower than that of MSSP (q 12 h, 52.27; q 8 h, 52.27) (p = 0.7710). These findings show the moderately phenotypic cotrimazole susceptibilities of both MRSP and MSSP. Further study is required to develop clinical trials examining the use of cotrimazole in dogs with pyoderma.

Evidence-based identification and characterization of methicillin-resistant Staphylococcusaureus isolated from subclinical mastitis in dairy buffaloes of Pakistan.

Methicillin-resistant Staphylococcus aureus (MRSA), affecting livestock and human beings, has become a global public health hazard with economic consequences. The current study was designed to investigate the prevailing MRSA-associated subclinical mastitis and associated risk factors in dairy buffaloes. The study also highlighted the genetic variations and in silico-based proteomic differences among MRSA isolates. Out of 516 milk samples, 45.93% (237/516) were found positive for subclinical mastitis, while the prevalence of S. aureus was recorded 56.12%. The methicillin resistance in S. aureus isolates was evaluated by oxacillin disc diffusion test and molecular identification of the mecA gene. The results revealed a phenotypic and molecular prevalence of MRSA at 45.11% and 18.79%, respectively. The risk factor analysis revealed that among various assumed risk factors, parity, milking hygiene, milker care during milking, milk yield, housing system, and floor type were significantly associated with subclinical mastitis in buffaloes. The sequencing and phylogenetic analysis showed no significant genetic variations among study isolates and depicted a high similarity with isolates from Africa, USA, India, Italy, Turkey, and Iran. The in-silico protein analysis showed that all sequences had the same protein motifs resembling penicillin protein 2a except Buff-13, whose protein structure resembles alpha-catenin-like protein hmp-1. The current study was the first report of the genotypic characterization and in silico protein analysis of MRSA from dairy buffaloes in Pakistan. The result highlighted the importance of antimicrobial resistance (AMR) and development of control strategies against MRSA infections.

Comparison of Different Phenotypic Methods of Detection of Methicillin-Resistant Staphylococcus aureus with Polymerase Chain Reaction

Objectives: The objective of the current study was to compare the diagnostic methods of Oxacillin Disk Diffusion, Cefoxitin Disk Diffusion, Oxacillin Resistance Screening Agar Base, and CHROM Agar MRSA with the gold-standard method of Polymerase Chain Reaction for detection of Methicillin-resistant Staphylococcus aureus.
 Methods: Two hundred pus samples were included in the study from which Staphylococcus strains were evaluated. The isolates of Staphylococcus aureus were subjected to the Oxacillin Disk Diffusion test, Cefoxitin Disk Diffusion test, Oxacillin Resistance Screening Agar Base, and CHROM Agar MRSA to detect MRSA with PCR, the reference standard. The diagnostic techniques were compared to their sensitivity, specificity, positive predictive, and negative predictive values.
 Results: The sensitivity of the Cefoxitin Disk Diffusion test was 100%, followed by CHROM Agar MRSA at 96.7%, Oxacillin Disk Diffusion at 90%, and Oxacillin Resistance Screening Agar Base at 86.7%. Most specific was the Cefoxitin Disk Diffusion test (99.4%), followed by Oxacillin Resistance Screening Agar Base (98.8%), CHROM Agar MRSA (97.7%), and the least specific was the Oxacillin Disk Diffusion test (96.5%).
 Conclusion: The Cefoxitin Disk Diffusion test was the most sensitive and specific of all four methods, next to the Polymerase Chain Reaction. However, future multicentric studies are recommended to test this method across all prevalent centers of methicillin resistance. J Microbiol Infect Dis 2022; 12(3):116-126.

Sources and Risk factors of a Novel MRSA Spa Type Circulating in Neonatal Intensive Care Unit

Background: Methicillin-Resistant Staphylococcus aureus is a major source of colonization and infection affecting premature neonates in Neonatal Intensive Care Unit. Objectives: to determine the prevalence, risk factors, and resistant profiles in neonates, their mothers, and healthcare personnel. In addition, to confirm the clonal similarity of neonatal Methicillin-Resistant S. aureus to their mothers or healthcare personnel. Materials and Methods: Samples were screened and identified for Methicillin-Resistant S. aureus colonization by gram stain, biochemical tests, oxacillin disk diffusion test, and resistance pattern. PCR was used to detect the presence of nuc and mecA gene. DNA fingerprinting was conducted using a standard spa typing technique. Results: Healthcare personnel colonization was high (27.9%) compared to neonatal and maternal colonization (15F%, 9.7%, respectively) and was found to be the only significant risk factor for neonatal MRSA colonization. Fifteen different spa types were identified and the novel t12492 was predominant among neonates and was reported for the first time. There was no demonstrated distribution correlation with sources of colonization. Colonization appeared to originate from multiple sources. Conclusion: Findings suggest conducting periodical molecular investigations of colonization of MRSA in healthcare personnel. Surveillance, molecular analysis of strains, reinforcement of an inclusive infection control program and antibiotic control could be useful in preventing MRSA transmission.

Isolation, characterisation and antibiotic susceptibility of staphylococcal isolates with special reference to methicillin-resistant Staphylococcus aureus from the anterior nares of healthcare workers in A tertiary healthcare centre

Background: Staphylococcus aureus (S. aureus) and its resistant form methicillinresistant S. aureus (MRSA) is one of the most common nosocomial pathogens causing a wide range of infections in humans. The anterior nares are the main ecological niche for S. aureus. Nasal carriage of S. aureus acts as an important reservoir of infection among the colonised healthcare workers and they transmit the infection to the community. The aim of the present study was to estimate the nasal colonisation of S. aureus (with special reference to MRSA) in healthcare workers (doctors and nursing staff) and its antibiotic susceptibility pattern. Methods: A descriptive study was planned in the Department of Microbiology, JLN Medical College, Ajmer (Rajasthan, India) after due approval from the institutional ethics committee. A total of 170 healthcare workers of either sex aged between 18 to 60 years were screened for S. aureus. Identification was done using standard microbiological techniques, by studying their morphology, colony and biochemical characteristics. MRSA was detected by cefoxitin disc diffusion test, oxacillin disc diffusion test, minimum inhibitory concentration (MIC) of oxacillin by E-test and oxacillin screen agar test. The observations were described in proportions and Chisquared test was used to find independence. Statistical significance was considered at 5 %. Results: Among 170 samples, 159 (93.53 %) samples (50 doctors and 109 nursing staff) had staphylococci colonisation. Among these 159 isolates, 34 (21.38 %) were S. aureus. Further, 8 (5.03 %) S. aureus isolates were resistant to both cefoxitin and oxacillin and had oxacillin MIC values ≥ 4 µg/mL and were considered MRSA. All the MRSA were detected in the nursing staff (males: 5.50 %, females: 1.83 %). All S. aureus and MRSA isolates were found sensitive to linezolid, vancomycin and mupirocin (minimum inhibitory concentration ≤ 4 µg/mL). Conclusion: Screening and treatment of healthcare workers colonised with MRSA should be an important component of hospital infection control policy. These measures will prevent spread of infection to patients and the community and thereby reduce the morbidity, mortality and healthcare costs associated with nosocomial infections.

Epidemiology and antibiotic succeptibility profile of methicillin sensitive Staphylococcus aureus among livestock and pet animals

Staphylococcus aureus is an important zoonotic pathogen that is responsible for a variety of infectious diseases in humans and animals. The present study was designed to check the prevalence and antimicrobial resistance of MSSA from three different animal origins (bovine, caprine and pet). A total of n= 450 samples (150 each source) were collected from bovine, caprine and pets. Collected samples were subjected to S. aurues identification by microbiological examination and confirmed S. aurues isolates were put to oxacillin disk diffusion test to declare them MSSA. The MSSA confirmed isolates were subjected to various antibiotics for susceptibility profiling using Kirby Baur Disk Diffusion test. The present study found higher prevalence of MSSA from caprine origin (goat 83.33%) as compared to pet (cat 69.33%; dog 65.33%) and bovine origin (buffalo 26.66%; cattle 31.66%). The in-vitro findings of current study revealed oxytetracycline and gentamicin presented 100% efficacy against MSSA of all origins while the vancomycin presented >35%, >40% and > 65% resistance against MSSA isolated from bovine, caprine and pet origin respectively. However, ciprofloxacin was equally effective (50%) against MSSA from buffalo and cattle while >80% efficacy was noted against MSSA from cat and dogs. Linezolid and amoxicillin+ clavulanic acid were 77.78% and 66.67% sensitive to MSSA isolates from caprine milk. The present study found higher prevalence of MSSA from bovine, caprine and pet isolates with diversified pattern of susceptibility of different antibiotics from all sources.

Molecular detection and genotypic characterisation of Streptococcus pneumoniae isolated from children in Malaysia

ABSTRACTStreptococcus pneumoniae (S. pneumoniae) is one of the main causative agents of pneumococcal diseases. To date, more than 90 distinct serotypes have been identified. Implementation of vaccines has caused a drastic reduction in vaccine-serotype pneumococcal diseases but increase in cases due to non-vaccine serotype has been observed in Malaysia. However, further investigation on different serotype incidence in Malaysia is needed and the rate of pneumococcal vaccination for new-born babies in Malaysia remains low. The recent emergence of drug-resistant S. pneumoniae (DRSP) has also been a global concern, especially penicillin resistance. This study determined the serotypes of S. pneumoniae strains (n = 95) isolated from nasopharyngeal specimens from children admitted to UMMC from 2013 to 2015. In accordance with previous studies, PCR result showed 40% of NT isolates were successfully typed as 3 less common serotypes, namely 9N/L, 17A, and 23B. The repetitive-element PCR (REP-PCR) result revealed genetic variations among the strains whereby five major clusters were observed at the similarity of 80% by clustering analysis based on fingerprint data. Penicillin-binding proteins (pbps) of selected isolates were studied by PCR and sequencing. Three strains with ≤19-mm diameter zone for Oxacillin Disc Diffusion (ODD) test previously were recorded to have mutation on all pbp1a, pbp2b, and pbp2x with MIC of 4 µg/ml, which were penicillin-intermediate resistance according to the CLSI breakpoints.

Epidemiology, Clinical Characteristics, and Antimicrobial Susceptibility Profiles of Human Clinical Isolates of Staphylococcus intermedius Group.

The veterinary pathogens in the Staphylococcus intermedius group (SIG) are increasingly recognized as causes of human infection. Shared features between SIG and Staphylococcus aureus may result in the misidentification of SIG in human clinical cultures. This study examined the clinical and microbiological characteristics of isolates recovered at a tertiary-care academic medical center. From 2013 to 2015, 81 SIG isolates were recovered from 62 patients. Patients were commonly ≥50 years old, diabetic, and/or immunocompromised. Documentation of dog exposure in the electronic medical record was not common. Of the 81 SIG isolates, common sites of isolation included 37 (46%) isolates from wound cultures and 17 (21%) isolates from respiratory specimens. Although less common, 10 (12%) bloodstream infections were documented in 7 unique patients. The majority of SIG (65%) isolates were obtained from polymicrobial cultures. In comparison to S. aureus isolates from the same time period, significant differences were noted in proportion of SIG isolates that were susceptible to doxycycline (74% versus 97%, respectively; P < 0.001), trimethoprim-sulfamethoxazole (65% versus 97%, respectively; P < 0.001), and ciprofloxacin (78% versus 59%, respectively; P < 0.01). Methicillin resistance (MR) was detected in 12 (15%) of 81 SIG isolates. All MR isolates detected by an oxacillin disk diffusion test would have been misclassified as methicillin susceptible using a cefoxitin disk diffusion test. Thus, SIG is recovered from human clinical specimens, and distinction of SIG from S. aureus is critical for the accurate characterization of MR status in these isolates.

Phenotypic and genotypic characterization of antibiotic resistance of methicillin-resistant Staphylococcus aureus isolated from hospital food

BackgroundPathogenic biotypes of the Methicillin-resistant Staphylococcus aureus (MRSA) strains are considered to be one of the major cause of food-borne diseases in hospitals. The present investigation was done to study the pattern of antibiotic resistance and prevalence of antibiotic resistance genes of different biotypes of the MRSA strains isolated from various types of hospital food samples.MethodsFour-hundred and eighty-five raw and cooked hospital food samples were cultured and MRSA strains were identified using the oxacillin and cefoxitin disk diffusion tests and mecA-based PCR amplification. Isolated strains were subjected to biotyping and their antibiotic resistance patterns were analyzed using the disk diffusion and PCR methods.ResultsPrevalence of S. aureus and MRSA were 9.69 and 7.62%, respectively. Meat and chicken barbecues had the highest prevalence of MRSA. Prevalence of bovine, ovine, poultry and human-based biotypes in the MRSA strains were 8.10, 8.10, 32.43 and 48.64%, respectively. All of the MRSA strains recovered from soup, salad and rice samples were related to human-based biotypes. MRSA strains harbored the highest prevalence of resistance against penicillin (100%), ceftaroline (100%), tetracycline (100%), erythromycin (89.18%) and trimethoprim-sulfamethoxazole (83.78%). TetK (72.97%), ermA (72.97%), msrA (64.86%) and aacA-D (62.16%) were the most commonly detected antibiotic resistance genes.ConclusionsPattern of antibiotic resistance and also distribution of antibiotic resistance genes were related to the biotype of MRSA strains. Presence of multi-drug resistance and also simultaneous presence of several antibiotic resistance genes in some MRSA isolates showed an important public health issue Further researches are required to found additional epidemiological aspects of the MRSA strains in hospital food samples.

PERFORMANCE OF CHROM AGAR MEDIUM AND CONVENTIONAL METHODS FOR DETECTION OF METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS.

Background:- Methicillin resistant Staphylococcus aureus (MRSA) are responsible for hospital and community acquired infections. There are many laboratory methods for detection of MRSA. Chromogenic media have been used for the last few years for the quick detection of MRSA. Objective:- Aim of this study was to compare the performance of conventional methods and chromogenic media for the detection of MRSA in a tertiary care hospital. Material and method: - 200 consecutive isolates of S. aureus confirmed by conventional methods, collected in a tertiary care hospital were used for this study. Cefoxitin and oxacillin disc diffusion test used as conventional methods and Chromogenic media i.e. oxacillin resistant screen agar base (ORSAB) was used for detection of methicillin resistant Staphylococcus aureus. All confirmed MRSA were checked by gold standard mecA base PCR method. Result: - Out of 200 isolates of Staphylococcus aureus, 50,52 and 47 strains were MRSA by Cefoxitin disc diffusion method, oxacillin disc diffusion method and oxacillin resistant screen agar base (ORSAB) method respectively. Specificity was 100%, 98.66%, 98.66% by Cefoxitin disc diffusion, oxacillin disc diffusion and ORSAB method respectively. Conclusion: - In conclusion, cefoxitin disc diffusion was the best for the phenotypic detection of MRSA because their sensitivity and specificity were better than oxacillin and ORSAB.

Evaluation of MRSA Chrome agar for the detection of methicillin resistant Staphylococcus aureus

The aim of this study was to evaluate the efficacy of MRSA Chrome agar to detect methicillin resistant Staphylococcus aureus (MRSA) and compare it with 1?g oxacillin disc diffusion tests and detection of mecA gene by PCR. A total 116 Staphylococcus aureus (S. aureus), isolated from various clinical samples, were obtained from three tertiary care hospitals of Dhaka city. S. aureus was identified by colony characters, Gram stain and standard biochemical procedures. MRSA was detected by susceptibility to 1?g oxacillin disc, growth of denim blue color colonies of S. aureus on the Brilliance MRSA Chrome agar at 24 and 48 hours of incubation. PCR was performed for amplification of mecA gene as a gold standard method. Out of 116 isolated S. aureus, 33 (28.44%) were MRSA by oxacillin disc diffusion test where mecA gene was detected in 28 strains. On MRSA Chrome agar, 29 (25.0%) S. aureus produced denim blue colonies at 24 hours, of which 28 isolates possessed mecA gene. At 48 hours incubation, an additional 4 isolates yielded denim blue colonies from which mecA gene could not be identified. All the strains of S. aureus that produced denim blue colonies at 24 and 48 hours were resistant to oxacillin. The sensitivity, specificity and accuracy of oxacillin disc diffusion test were 100%, 94.31% and 95.68% and Chrome agar at 24 hours were 100%, 98.86% and 99.13% respectively. Thus MRSA Chrome agar could be good choice in clinical microbiology laboratory for rapid and accurate identification of MRSA. DOI: http://dx.doi.org/10.3329/imcj.v7i1.17697 Ibrahim Med. Coll. J. 2013; 7(1): 1-4

English

The emergence of heterogeneous populations of methicillin-resistant&nbsp;Staphylococcus aureus&nbsp;(MRSA) causes major problems in routine screening for MRSA. Cefoxitin is a potent inducer of the&nbsp;mecA&nbsp;regulatory system and can be use for detection of heterogeneous populations of MRSA. Detection of the&nbsp;mecA&nbsp;gene by PCR was considered to be the &ldquo;gold standard&rdquo;. In this study we determined the sensitivity and specificity of the oxacillin disk diffusion test, cefoxitin disk diffusion test and oxacillin agar screening for detection of MRSA. A total of 124 non-duplicate isolates of&nbsp;S. aureus&nbsp;were included in the study. Methicillin resistance was measured using oxacillin (1&mu;g) and cefoxitin(30&mu;g) disc diffusion method and oxacillin agar screening test (6 mg/ml oxacillin) according to CLSI guideline and PCR for the&nbsp;mecA gene. Compared with the molecular detection of methicillin resistance the overall sensitivities and specificities of the phenotypic tests for cefoxitin disc diffusion were 100%, for oxacillin disc diffusion were 91.7 and 92.8% and for oxacillin agar screening were 95 and 95.5%, respectively. We concluded that in the absence of availability of molecular biology techniques, the cefoxitin disc was the best detector of methicillin resistance in&nbsp;S. aureus&nbsp;related to the other phenotypic tests. &nbsp; Key words:&nbsp;Methicillin-resistant&nbsp;Staphylococcus aureus, mecA, Cefoxitin, polymerized chain reaction

Identification of a Novel Lytic Bacteriophage Obtained from Clinical MRSA Isolates and Evaluation of Its Antibacterial Activity

Multidrug-resistant bacteria particularly MRSA is well known as a worldwide problem. Since the rate of development of novel antimicrobial agents has been slowed down during the last years, there have been a need for the exploration of alternative solutions for the treatment of resistant bacterial infections. Treatment of infections by bacteriophages (phages) that specifically kill the infecting pathogen, i.e. by the process known as phage therapy, is considered as a possible approach to treat multidrug resistant bacteria. Phage treatment has also been considered to treat Staphylococcus aureus infections. This study was aimed to evaluate the antibacterial and cytotoxic activities of a new lytic phage obtained from clinical MRSA strains. This lytic phage named as f LizAnk was obtained during the phage infectivity studies performed with 13 lysogenic phages against MRSA strains. The antibacterial activity of the f LizAnk phage was determined in vitro in BHI (Brain Heart Infusion) and LB (Leuria Bertani) broths and the in vivo antibacterial activity against MRSA strains and possible cytotoxic effect against mammalian cells were tested on fibroblastic cell cultures (3T3). This study was conducted using 20 MRSA strains isolated from hospitalized patients. Identification of the isolates was performed by conventional methods and methicillin resistance was detected with oxacillin disk diffusion test and mecA gene detection by PCR. The method described by Kaneko et al. [Biosci Biotechnol Biochem 1997; 61(11): 1960-2] was used with some modifications, for induction and isolation of the phages. In vitro studies indicated that this phage killed the six different MRSA strains (in 107 cfu/ml concentrations) in 8 hours, and this powerful lytic effect was similar in both of the liquid media. In vivo studies were performed by using cell cultures prepared in microplates, and the wells have been inoculated with only phage, phage + MRSA mixture, and only MRSA. The cells were then evaluated microscopically as well as by MTT assay which detected alive cells colorimetrically, at 2nd and 24th hours. In our study, the f LizAnk phage did not cause any toxic effect on fibroblast cell cultures, in addition it was observed that the antibacterial effect of the phage against MRSA has proceeded in the cell culture. In conclusion, since the fLizAnk phage described in this study exhibited strong antibacterial activity against MRSA strains and no cytotoxic effect was detected against mammalian cells, it might be safely used alone or in a phage cocktail to treat skin infection caused by MRSA.

Comparison of Cefoxitin and Oxacillin disc diffusion test for the detection of mecA mediated methicillin resistance in Staphylococcus aureus

The study was designed to evaluate the efficacy of cefoxitin disc diffusion test to detect methicillin resistance in Staphylococcus aureus and compare it with oxacillin disc diffusion test and detection of mecA gene by PCR. A total 116 S. aureus were isolated from clinical samples, collected from SSMC&amp;MH, BIRDEM and NMC hospital, and was isolated by culture and identified by standard laboratory procedure. Antibiotic susceptibility testing was performed by oxacillin (1µg) and cefoxitin (30 µg) discs. PCR for amplification of mecA gene was performed as a gold standard method. Out of 116 isolates, 28 were PCR positive, 33 and 31 were oxacillin and cefoxitin resistant respectively. The sensitivity and specificity for the detection of MRSA was 100% and 94.31%inoxacillin disc diffusion test, and 96.42% and 95.45%in cefoxitin disc diffusion test respectively. Specificity is higher (95.45%) in cefoxitin disc diffusion test than oxacillin disc diffusion test in the detection of MRSA. Use of disc diffusion tests for both oxacillin and cefoxitin can help in more accurate prediction of methicillin resistance than single test, especially in centers which are not equipped to carry out more sophisticated tests for the detection of MRSA.DOI: http://dx.doi.org/10.3329/bjmm.v7i1.19314 Bangladesh J Med Microbiol 2013; 07(01): 7-10

Detecting methicillin resistance in Staphylococcus aureus: comparison of different phenotypic methods and the polymerase chain reaction

Cefoxitin is a more potent inducer of the mecA regulatory system compared to oxacillin in Staphylococcus aureus. It has been recommended for the detection of methicillin-resistant Staphylococcus aureus (MRSA) when using disk-diffusion testing. The aim of this study is to compare the results of cefoxitin and oxacillin disk-diffusion methods against the polymerase chain reaction (PCR) for the detection of MRSA. A total of 75 strains of S. aureus isolated from different clinical specimens (e.g., pus, blood, wound swabs, tracheal aspirates, eye swabs and cerebrospinal fluid [CSF]) were screened for methicillin resistance by PCR and cefoxitin and oxacillin disk-diffusion tests. The antibiotic susceptibility testing was performed by the Baur disk-diffusion method using oxacillin (1 |ig) and cefoxitin (30 |ig) disks on Mueller Hinton agar (MHA), and zone diameters as recommended by CLSIs were read at 18 h and 24 h. All MRSA isolates detected phenotypically were confirmed by PCR for the amplification of mecA and nucA genes. Of the 75 isolates screened, 27 were resistant to oxacillin, 30 were resistant to cefoxitin using the disk-diffusion method, while 30 isolates were confirmed as MRSA by PCR. Performing the cefoxitin disk-diffusion method using a 30 | g disk could be a reliable and more accurate method to detect methicillin resistance in S. aureus strains in situations where mecA PCR cannot be performed.

Health Care Workers’ Mobile Phones: A Potential Cause of Microbial Cross-Contamination Between Hospitals and Community

This study evaluated the microbial contamination of health care workers’ (HCWs) mobile phones. The study was conducted at a secondary referral hospital in July 2010. Samples were taken from all surfaces of the mobile phones using a sterile swab, and incubated on Brain Heart Infusion agar at 37.5°C for 24 hr. Any isolated microorganisms were grown aerobically on 5% sheep blood agar and eosin methylene-blue agar medium at 37.5°C for 24–48 hr. The Sceptor microdilution system was used to identify the microorganisms, together with conventional methods. The oxacillin disc diffusion test and double-disc synergy test were used to identify methicillin-resistant Staphylococcus aureus (MRSA) and expanded-spectrum beta-lactamase (ESBL)-producing Gram-negative bacilli, respectively. The mobile phones were also categorized according to whether the HCWs used them in the intensive care unit (ICU). Overall, 183 mobile phones were screened: 94 (51.4%) from nurses, 32 (17.5%) from laboratory workers, and 57 (31.1%) from health care staff. In total, 179 (97.8%) culture-positive specimens were isolated from the 183 mobile phones, including 17 (9.5%) MRSA and 20 (11.2%) ESBL-producing Escherichia coli, which can cause nosocomial infections. No statistical difference was observed in the recovery of MRSA (p = 0.3) and ESBL-producing E. coli (p = 0.6) between the HCW groups. Forty-four (24.6%) of the 179 specimens were isolated from mobile phones of ICU workers, including two MRSA and nine ESBL-producing E. coli. A significant (p = 0.02) difference was detected in the isolation of ESBL-producing E. coli between ICU workers and non-ICU workers. HCWs’ mobile phones are potential vectors for transferring nosocomial pathogens between HCWs, patients, and the community.

Analysis of cefoxitin-sensitive, penicillin-resistant Staphylococcus tested by disk diffusion method

Objective To evalue the ability of detecting the resistance of cefoxitin-sensitive,penicillin-resistant Staphylococcus by different methods and analyze the antibiotic susceptibility spectrum of coagulase-negative Staphylococcus which are non-mecA-mediated oxacillin resistance. Methods All the isolates were collected from Huashan hospital between 2007 and 2009. The isolates were recovered from various clinical sources, including respiratory tract, urine, secretion and sterile fluids samples. The oxacillin susceptibility of Staphylococcus aureus was determined by cefoxitin disk diffusion test, cefoxitin MIC test,oxacillin disk diffusion test and oxacillin MIC test Likewise, the oxacillin susceptibility of coagulasenegative Staphylococcus was determined by cefoxitin disk diffusion test and oxacillin MIC test. All the isolates with sensitive to cefoxitin were screened for the mec A gene by PCR Finally, the MIC of non-mecA-mediated oxacillin-resistant Staphylococcus were determined. Results Among 255 cefoxitin disk diffusion test sensitive and penicillin-resistant Staphylococcus aureus, 6 isolates were intermediated to oxacillin and 4 were resistant by oxacillin disk diffusion test, but all the isolates were sensitive by the cefoxitin disk diffusion test,cefoxitin MIC test and oxacillin MIC test. Among 75 cefoxitin disk diffusion test sensitive and penicillin-resistant coagulase-negative Staphylococcus, 16 isolates were resistant to oxacillin by oxacillin MIC method and 4 carried mecA gene. Among 12 non-mecA-mediated oxacillin-resistant Staphylococcus, the susceptible isolates of gentamicin is 10, clindamycin is 8, ciprofloxacin is 11, erythrornycin is 6, trimethoprim/sulfamethoxazo]e is 11 ,and cephalosporins, teicoplaninl, vancomycin, piperacillin/tazobactam, tetracycline are all 12. Conclusions The cefoxitin disk diffusion test can reliably predict mecA-mediated oxacillin resistant Staphylococcus aureus. It would be best to combine cefoxitin disk diffusion test and oxacillin MIC test to improve accuracy of detection of mecA-mediated oxacillin resistant coagulase-negative Staphylococcus.Furthermore, infections due to the non-mecA-mediated oxacillin resistant coagulase-negative Staphylococcus can be treated by penicillinase-stable penicillins, β-lactam/β-lactam inhibitor combinations, relevant cephems and carbapenems. Key words: Staphylococcus aureus; Penicillin resistance; Cefoxitin; Disk diffusion antimicrobial tests

Avaliação de métodos comumente usados em laboratórios para a determinação da suscetibilidade à oxacilina entre amostras de Staphylococcus sp, isoladas de um hospital de Vitória, Estado do Espírito Santo

The genus Staphylococcus is of great importance because of its high prevalence in hospital infections and because it presents high rates of resistance to oxacillin and other antimicrobials. Thus, evaluation of the accuracy of the phenotypic methods that are used to determine the profile of antimicrobial resistance is essential to ensure that the most appropriate therapy is chosen. One hundred and fourteen strains of Staphylococcus sp (53 S. aureus and 61 CNS) were used to evaluate the accuracy of the methods of disk diffusion, agar microdilution, oxacillin screening agar and automated systems, in comparison with PCR for investigating resistance to oxacillin. The mecA gene was detected in 48 strains (42.1%), and 27 strains (23.7%) showed discrepant results in at least one of the methods (74.1% of CNS, 25.9% of S. aureus). For S. aureus, with the exception of the Microscan Walkaway, all the methods showed 100% specificity and sensitivity. In relation to CNS, the automated system and cefoxitin disk had lower accuracy. Use of two methods should be the best option for improved accuracy, especially when the diagnostic laboratory only uses an automated system or oxacillin disk diffusion test. Combination of these methods with others presented almost 100% sensitivity and specificity in our study.