BRD4 directs hematopoietic stem cell development and modulates macrophage inflammatory responses.

BRD4 is a BET family protein that binds acetylated histones and regulates transcription. BET/BRD4 inhibitors block blood cancer growth and inflammation and serve as a new therapeutic strategy. However, the biological role of BRD4 in normal hematopoiesis and inflammation is not fully understood. Analysis of Brd4 conditional knockout (KO) mice showed that BRD4 is required for hematopoietic stem cell expansion and progenitor development. Nevertheless, BRD4 played limited roles in macrophage development and inflammatory response to LPS ChIP-seq analysis showed that despite its limited importance, BRD4 broadly occupied the macrophage genome and participated in super-enhancer (SE) formation. Although BRD4 is critical for SE formation in cancer, BRD4 was not required for macrophage SEs, as KO macrophages created alternate, BRD4-less SEs that compensated BRD4 loss. This and additional mechanisms led to the retention of inflammatory responses in macrophages. Our results illustrate a context-dependent role of BRD4 and plasticity of epigenetic regulation.

Shifts in the Molecular Epidemiology of Campylobacter jejuni Infections in a Sentinel Region of New Zealand following Implementation of Food Safety Interventions by the Poultry Industry.

In 2006, New Zealand had the highest notification rate of campylobacteriosis in the world, and poultry was considered the leading source of campylobacteriosis. Implementation of food safety interventions by the poultry industry led to a decrease in the campylobacteriosis notification rate. The aim is to examine the impact of targeted food safety interventions implemented by the New Zealand poultry industry on the source attribution of Campylobacter jejuni infections in a sentinel region. Campylobacter jejuni isolates collected from the Manawatu region of New Zealand between 2005 and 2007 ("before intervention") and 2008 and 2015 ("after intervention") from human clinical cases, chicken meat, ruminant feces, environmental water, and wild bird sources were subtyped by multilocus sequence typing. Viable counts of Campylobacter spp. from carcasses were analyzed using a zero-inflated Poisson regression model. In the period before intervention, sequence type 474 (ST-474) was the most common sequence type (ST) recovered from human cases, accounting for 28.2% of the isolates. After intervention, the proportion of human cases positive for ST-474 reduced to 9.3%. Modeling indicated that chicken meat, primarily from one supplier, was the main source of C. jejuni infection in the Manawatu region before intervention. However, after intervention poultry collectively had a similar attribution to ruminants, but more human cases were attributed to ruminants than any single chicken supplier. Viable counts on carcasses were lower in all poultry suppliers after intervention. This study provides evidence of changes in the source attribution of campylobacteriosis following targeted food safety interventions in one sector of the food supply chain.IMPORTANCE This study provides a unique insight into the effects of food safety interventions implemented in one sector of the food industry on the transmission routes of a major foodborne agent. Following the implementation of food safety interventions by the poultry industry, shifts in the molecular epidemiology of Campylobacter jejuni infections in a sentinel region of New Zealand were observed. Targeted interventions to reduce disease incidence are effective but require continued surveillance and analysis to indicate where further interventions may be beneficial.

Short communication: Bovine mastitis caused by a multidrug-resistant, mcr-1-positive (colistin-resistant), extended-spectrum ß-lactamase-producing Escherichia coli clone on a Greek dairy farm.

We performed a survey aimed at analyzing milk samples collected from cows with mastitis for the presence of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli. Single-quarter mastitic milk samples obtained from 400 cows in 23 Greek dairy herds with a history of E. coli mastitis were processed for the selective isolation of ESBL-producing E. coli. The antimicrobial susceptibility of the ESBL-producing isolates was analyzed using agar disk diffusion, and minimum inhibitory concentrations of colistin were determined by broth microdilution. We used PCR followed by DNA sequencing to characterize the ß-lactamases and mcr-1 (colistin resistance) genes, and for phylotyping and multilocus sequence typing. We found a total of 89/400 (22.25%) E. coli isolates from 12/23 (52%) farms. Six isolates originating from 6 cows on a single farm were ESBL producers and were resistant to cefquinome, amoxicillin-clavulanic acid, aztreonam, ampicillin, and colistin. Five of these isolates were resistant to trimethoprim-sulfamethoxazole, and 5 to streptomycin. The 6 ESBL producers were mcr-1-positive and carried blaTEM-1 genes; 3 also carried blaCTX-M genes, and 3 carried blaSHV genes. All of the ESBL producers belonged to phylogroup A, multilocus sequence type ST666 (n = 5), or a single locus variant of ST666 (n = 1). To our knowledge, this is the first report of endemic bovine mastitis caused by mcr-1-positive, ESBL-producing E. coli. These results highlight the value of active surveillance of antimicrobial resistance not commonly tested by diagnostic laboratories for the early detection of novel resistant strains.

Coreceptor gene imprinting governs thymocyte lineage fate.

Immature thymocytes are bipotential cells that are signalled during positive selection to become either helper- or cytotoxic-lineage T cells. By tracking expression of lineage determining transcription factors during positive selection, we now report that the Cd8 coreceptor gene locus co-opts any coreceptor protein encoded within it to induce thymocytes to express the cytotoxic-lineage factor Runx3 and to adopt the cytotoxic-lineage fate, findings we refer to as 'coreceptor gene imprinting'. Specifically, encoding CD4 proteins in the endogenous Cd8 gene locus caused major histocompatibility complex class II-specific thymocytes to express Runx3 during positive selection and to differentiate into CD4(+) cytotoxic-lineage T cells. Our findings further indicate that coreceptor gene imprinting derives from the dynamic regulation of specific cis Cd8 gene enhancer elements by positive selection signals in the thymus. Thus, for coreceptor-dependent thymocytes, lineage fate is determined by Cd4 and Cd8 coreceptor gene loci and not by the specificity of T-cell antigen receptor/coreceptor signalling. This study identifies coreceptor gene imprinting as a critical determinant of lineage fate determination in the thymus.

Molecular Epidemiology of Campylobacter coli Strains Isolated from Different Sources in New Zealand between 2005 and 2014.

UNLABELLED: Campylobacteriosis is one of the most important foodborne diseases worldwide and a significant health burden in New Zealand. Campylobacter jejuni is the predominant species worldwide, accounting for approximately 90% of human cases, followed by Campylobacter coli Most studies in New Zealand have focused on C. jejuni; hence, the impact of C. coli strains on human health is not well understood. The aim of this study was to genotype C. coli isolates collected in the Manawatu region of New Zealand from clinical cases, fresh poultry meat, ruminant feces, and environmental water sources, between 2005 and 2014, to study their population structure and estimate the contribution of each source to the burden of human disease. Campylobacter isolates were identified by PCR and typed by multilocus sequence typing. C. coli accounted for 2.9% (n = 47/1,601) of Campylobacter isolates from human clinical cases, 9.6% (n = 108/1,123) from poultry, 13.4% (n = 49/364) from ruminants, and 6.4% (n = 11/171) from water. Molecular subtyping revealed 27 different sequence types (STs), of which 18 belonged to clonal complex ST-828. ST-1581 was the most prevalent C. coli sequence type isolated from both human cases (n = 12/47) and poultry (n = 44/110). When classified using cladistics, all sequence types belonged to clade 1 except ST-7774, which belonged to clade 2. ST-854, ST-1590, and ST-4009 were isolated only from human cases and fresh poultry, while ST-3232 was isolated only from human cases and ruminant sources. Modeling indicated ruminants and poultry as the main sources of C. coli human infection. IMPORTANCE: We performed a molecular epidemiological study of Campylobacter coli infection in New Zealand, one of few such studies globally. This study analyzed the population genetic structure of the bacterium and included a probabilistic source attribution model covering different animal and water sources. The results are discussed in a global context.

An ectopic CTCF-dependent transcriptional insulator influences the choice of Vß gene segments for VDJ recombination at TCRß locus.

Insulators regulate transcription as they modulate the interactions between enhancers and promoters by organizing the chromatin into distinct domains. To gain better understanding of the nature of chromatin domains defined by insulators, we analyzed the ability of an insulator to interfere in VDJ recombination, a process that is critically dependent on long-range interactions between diverse types of cis-acting DNA elements. A well-established CTCF-dependent transcriptional insulator, H19 imprint control region (H19-ICR), was inserted in the mouse TCRß locus by genetic manipulation. Analysis of the mutant mice demonstrated that the insulator retains its CTCF and position-dependent enhancer-blocking potential in this heterologous context in vivo. Remarkably, the inserted H19-ICR appears to have the ability to modulate cis-DNA interactions between recombination signal sequence elements of the TCRß locus leading to a dramatically altered usage of Vß segments for Vß-to-DßJß recombination in the mutant mice. This reveals a novel ability of CTCF to govern long range cis-DNA interactions other than enhancer-promoter interactions and suggests that CTCF-dependent insulators may play a diverse and complex role in genome organization beyond transcriptional control. Our functional analysis of mutated TCRß locus supports the emerging role of CTCF in governing VDJ recombination.

Targeted Sos1 deletion reveals its critical role in early T-cell development.

Activation of the small G protein Ras is required for thymocyte differentiation. In thymocytes, Ras is activated by the Ras guanine exchange factors (RasGEFs) Sos1, Sos2, and RasGRP1. We report the development of a floxed allele of sos1 to assess the role of Sos1 during thymocyte development. Sos1 was required for pre-T-cell receptor (pre-TCR)- but not TCR-stimulated developmental signals. Sos1 deletion led to a partial block at the DN-to-DP transition. Sos1-deficient thymocytes showed reduced pre-TCR-stimulated proliferation, differentiation, and ERK phosphorylation. In contrast, TCR-stimulated positive selection, and negative selection under strong stimulatory conditions, remained intact in Sos1-deficient mice. Comparison of RasGEF expression at different developmental stages showed that relative to Sos2 and RasGRP1, Sos1 is most abundant in DN thymocytes, but least abundant in DP thymocytes. These data reveal that Sos1 is uniquely positioned to affect signal transduction early in thymocyte development.

Genomic epidemiology of bovine mastitis-causing Staphylococcus aureus in New Zealand.

We analysed the genomes of 188 bovine-mastitis-causing S. aureus isolates obtained over a 17-year period from more than 65 dairy farms across New Zealand. The analysis revealed a unique pattern of dominance over the entire period of study, of clonal complex 1, sequence type 1 (CC1/ST1), which accounted for ∼75% of the isolates. CC1/ST1 was also the commonest lineage infecting humans in New Zealand in the same period, but most bovine CC1/ST1 analysed in this study carried the genes coding for the bovine-adaptive bicomponent leucocidin lukF and lukM and lacked the corresponding human-adaptive lukF-PV and lukS-PV genes. Typical ruminant-associated lineages, such as ST97, ST151 and CC133 were also observed. Cluster analyses of the core and accessory genomes revealed genomic segregations according to the CCs, but lack of segregation based on the geographical location or collection year, suggesting a stable population in space and time. To our knowledge, this is the first identification of genomic markers of host adaptation to cattle in S. aureus CC1/ST1, a lineage commonly associated with humans, worldwide. The temporal clonal stability observed would enable the development of a S. aureus vaccine for New Zealand cattle, which is unlikely to undergo substantial reduction of efficacy due to clonal drifts or shifts.

Staphylococcus microti Strains Isolated from an Italian Mediterranean Buffalo Herd.

S. microti is a new species among non-aureus staphylococci (NAS) frequently found in bovine milk samples and associated with subclinical mastitis (SCM). The aim of this study was to analyze the presence of S. microti in 200 composite milk samples and 104 milking parlor surface swabs collected at a buffalo farm in Southern Italy to define its presence in milk and a milking parlor environment. The samples were inoculated onto different agar plates, and the isolates were identified by MALDI-TOF MS. The strains identified as S. microti (54/304 samples, 17.8%) were collected, and their purified genomic DNA was subjected to PCR amplification and whole 16S rRNA gene sequencing. Furthermore, their phenotypic resistance profiles were evaluated by a disk diffusion method, and the genotypic characterization of the tetracycline resistance was performed for the tetM and tetK genes by multiplex PCR. Four and forty-seven S. microti isolates from milk samples of lactating animals with subclinical mastitis (SCM) and intramammary infection (IMI), respectively, and three isolates from milking parlor surfaces were recovered. The genomic DNA was purified from the bacterial isolates, and the amplification and sequencing of the 16S gene further supported the proteomic identification as S. microti. No clinical mastitis was detected in the herd during the study period. The antimicrobial susceptibility testing revealed a worrisome 100% resistance to tetracyclines, genotypically mediated by the tetM gene for all strains. This study highlights that S. microti may be commonly isolated from dairy buffalo milk and milking parlor equipment. Its association with SCM or IMI remains to be established.

The Rho guanine nucleotide exchange factor AKAP13 (BRX) is essential for cardiac development in mice.

A fundamental biologic principle is that diverse biologic signals are channeled through shared signaling cascades to regulate development. Large scaffold proteins that bind multiple proteins are capable of coordinating shared signaling pathways to provide specificity to activation of key developmental genes. Although much is known about transcription factors and target genes that regulate cardiomyocyte differentiation, less is known about scaffold proteins that couple signals at the cell surface to differentiation factors in developing heart cells. Here we show that AKAP13 (also known as Brx-1, AKAP-Lbc, and proto-Lbc), a unique protein kinase A-anchoring protein (AKAP) guanine nucleotide exchange region belonging to the Dbl family of oncogenes, is essential for cardiac development. Cardiomyocytes of Akap13-null mice had deficient sarcomere formation, and developing hearts were thin-walled and mice died at embryonic day 10.5-11.0. Disruption of Akap13 was accompanied by reduced expression of Mef2C. Consistent with a role of AKAP13 upstream of MEF2C, Akap13 siRNA led to a reduction in Mef2C mRNA, and overexpression of AKAP13 augmented MEF2C-dependent reporter activity. The results suggest that AKAP13 coordinates Galpha(12) and Rho signaling to an essential transcription program in developing cardiomyocytes.

Pancreatic candidiasis in a cat.

CASE SUMMARY: An 11-year-old female spayed Maine Coon cat was referred for assessment of hyporexia, weight loss, vomiting and diarrhoea. An abdominal ultrasound revealed an enlarged and hypoechoic pancreas containing two large fluid-filled structures. Fine-needle aspiration of the cyst-like structures was performed, and cytology revealed moderate-to-marked predominantly suppurative inflammation with yeast cells. Candida glabrata was cultured from the fluid. The patient was treated with oral itraconazole and the clinical signs resolved, although repeat abdominal ultrasound and cytology revealed persistence of the infected cyst-like structures. The patient remained asymptomatic for 8 months after the discontinuation of antifungal medications, despite the persistence of the pancreatic infection with C glabrata. RELEVANCE AND NOVEL INFORMATION: To our knowledge, this is the first report of pancreatic infection with Candida species in a cat, followed by a chronic subclinical infection persisting for at least 8 months after treatment discontinuation.

Molecular Evidence of Bartonella spp. in Rodents: A Study in Pianosa Island, Italy.

Wild rodents are reservoirs of several Bartonella species that cause human bartonellosis. The aim of this study was to assess the presence of Bartonella spp. DNA in wild rodents in Pianosa island, Italy. Rats (Rattus spp.; n = 15) and field mice (Apodemus spp.; n = 16) were captured and spleen DNA tested for the presence of Bartonella spp. by means of an initial screening using a qPCR amplifying a short segment of the 16S-23S rRNA gene intergenic transcribed spacer region (ITS, ~200 bp) followed by conventional PCR amplification of a longer ITS fragment (~600 bp) and of a citrate synthase (gltA, ~340 bp) gene segment. A total of 25 spleen DNA samples obtained from 31 rodent carcasses (81%) yielded positive qPCR results. Bartonella genus was confirmed by amplicon sequencing. By conventional PCR, eight out of 25 samples (32%) yielded bands on gels consistent with ITS segment, and 6/25 (24%) yielded bands consistent with the gltA locus. Amplicon sequencing identified B. henselae and B. coopersplainsensis in 1/25 (4%), and 4/25 (16%) samples, respectively. Moreover, 5/25 (20%) of Bartonella spp. positive samples showed gltA sequences with about 97% identity to B. grahamii. These results provide support to recently published observations suggesting that B. henselae circulates in wild rodent populations.

The multifunctional RNA-binding protein La is required for mouse development and for the establishment of embryonic stem cells.

The La protein is a target of autoantibodies in patients suffering from Sjögren's syndrome, systemic lupus erythematosus, and neonatal lupus. Ubiquitous in eukaryotes, La functions as a RNA-binding protein that promotes the maturation of tRNA precursors and other nascent transcripts synthesized by RNA polymerase III as well as other noncoding RNAs. La also associates with a class of mRNAs that encode ribosome subunits and precursors to snoRNAs involved in ribosome biogenesis. Thus, it was surprising that La is dispensable in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the organisms from which it has been characterized most extensively. To determine whether La is essential in mammals and if so, at which developmental stage it is required, mice were created with a disrupted La gene, and the offspring from La+/-intercrosses were analyzed. La-/- offspring were detected at the expected frequency among blastocysts prior to implantation, whereas no nullizygotes were detected after implantation, indicating that La is required early in development. Blastocysts derived from La+/- intercrosses yielded 38 La+/+ and La+/- embryonic stem (ES) cell lines but no La-/- ES cell lines, suggesting that La contributes a critical function toward the establishment or survival of ES cells. Consistent with this, La-/- blastocyst outgrowths revealed loss of the inner cell mass (ICM). The results indicate that in contrast to the situation in yeasts, La is essential in mammals and is one of a limited number of genes required as early as the development of the ICM.

Safety of a live attenuated Erysipelothrix rhusiopathiae vaccine for swine.

Infection with Erysipelothrix rhusiopathiae has a significant economic impact on pig production systems worldwide. Both inactivated and attenuated vaccines are available to prevent development of clinical signs of swine erysipelas. The ability of a live attenuated E. rhusiopathiae strain to become persistently established in pigs after intranasal exposure and its potential to cause clinical signs consistent with swine erysipelas after being administered directly into the nasopharynx of healthy pigs was evaluated. Five, E. rhusiopathiae-negative pigs were vaccinated by deep intranasal inoculation then followed for 14 days. Nasal swabs were collected daily for 5 days and clinical observations were made daily for 14 days post-vaccination. Nasal swabs were cultured for E. rhusiopathiae with the intent of back-passaging any recovered organisms into subsequent replicates. No organism was recovered from nasal swabs in the first vaccination replicate. A second replicate including 10 pigs was initiated and followed in an identical manner to that described above. Again, no E. rhusiopathiae was recovered from any pigs. No pigs in either replicate showed any signs of clinical swine erysipelas. The live attenuated E. rhusiopathiae strain evaluated in this study did not appear to become persistently established in pigs post-vaccination, did not cause any local or systemic signs consistent with swine erysipelas, and was therefore unlikely to revert to a virulent state when used in a field setting.

Multidrug resistance protein 4 protects bone marrow, thymus, spleen, and intestine from nucleotide analogue-induced damage.

Nucleoside-based analogues are mainstays in the treatment of cancer, viral infections, and inflammatory diseases. Recent studies showing that the ATP-binding cassette transporter, multidrug resistance protein 4, is able to efflux nucleoside and nucleotide analogues from transfected cells suggests that the pump may affect the efficacy of this class of agents. However, the in vivo pharmacologic functions of the pump are largely unexplored. Here, using Mrp4(-/-) mice as a model system, and the nucleotide analogue, 9'-(2'-phosphonylmethoxyethyl)-adenine (PMEA) as a probe, we investigate the ability of Mrp4 to function in vivo as an endogenous resistance factor. In the absence of alterations in plasma PMEA levels, Mrp4-null mice treated with PMEA exhibit increased lethality associated with marked toxicity in several tissues. Affected tissues include the bone marrow, spleen, thymus, and gastrointestinal tract. In addition, PMEA penetration into the brain is increased in Mrp4(-/-) mice. These findings indicate that Mrp4 is an endogenous resistance factor, and that the pump may be a component of the blood-brain barrier for nucleoside-based analogues. This is the first demonstration that an ATP-binding cassette transporter can affect in vivo tissue sensitivity towards this class of agents.

Carriage and population genetics of extended spectrum ß-lactamase-producing Escherichia coli in cats and dogs in New Zealand.

The incidence of infections with extended spectrum ß-lactamase producing Escherichia coli (ESBL-E) is increasing both in humans and animals. There is a paucity of data about the rate of faecal carriage of ESBL-E in pets. In this study, faecal swabs collected from 586 pets (225 cats; 361 dogs) in Auckland, New Zealand, were analysed for the presence of ESBL-E by culture, and a questionnaire was delivered to the owners. The ESBL-E were characterised and data elicited by the questionnaires were used for a multivariable analysis, to investigate the factors associated with faecal ESBL-E carriage. The prevalence of ESBL-E in faecal swabs was 6.4%. The ß-lactamase genes detected in the ESBL-E were the blaCTX-M-14 (n = 2) and blaCMY-2 (n = 34). Several isolates displayed multilocus sequence types (ST) associated with human and animal infections. Multiple isolates sharing the same ST displayed different antibiograms and ß-lactamase genes, reflecting horizontal gene transfer between and within ST. Variables independently associated with increased odds of ESBL-E carriage were: animal received systemic antimicrobial treatment in the six months before the sampling; presence of household members working in veterinary clinics; presence of household members travelling overseas in the six months before the sampling. We conclude that pets are colonised by ESBL-E which are genotypically similar to the bacteria found to infect humans and animals. The statistical analysis suggested a number of eco-epidemiological factors associated with ESBL-E carriage. In particular, they suggest veterinary clinics may represent hot-spots of antimicrobial resistance.

Inferences about the global population structures of Cryptosporidium parvum and Cryptosporidium hominis.

Cryptosporidium parvum and Cryptosporidium hominis are two related species of apicomplexan protozoa responsible for the majority of human cases of cryptosporidiosis. In spite of their considerable public health impact, little is known about the population structures of these species. In this study, a battery of C. parvum and C. hominis isolates from seven countries was genotyped using a nine-locus DNA subtyping scheme. To assess the existence of geographical partitions, the multilocus genotype data were mined using a cluster analysis based on the nearest-neighbor principle. Within each country, the population genetic structures were explored by combining diversity statistical tests, linkage disequilibrium, and eBURST analysis. For both parasite species, a quasi-complete phylogenetic segregation was observed among the countries. Cluster analysis accurately identified recently introduced isolates. Rather than conforming to a strict paradigm of either a clonal or a panmictic population structure, data are consistent with a flexible reproductive strategy characterized by the cooccurrence of both propagation patterns. The relative contribution of each pattern appears to vary between the regions, perhaps dependent on the prevailing ecological determinants of transmission.

Exposure to whole chicken carcasses may present a greater risk of campylobacteriosis compared to exposure to chicken drumsticks.

In New Zealand, the major risk factor for campylobacteriosis has been identified as poultry consumption. New Zealanders consume different types of chicken meat which undergo different processing before entering the retail chain. The manipulations and jointing of chicken carcasses into pieces and the subsequent processing and packaging have the potential to cross-contaminate and reshuffle bacterial pathogens among the different products sold. The aim of this study was to analyse: (a) the differences in the viable count and population genetic structure between Campylobacter isolated from chicken drumsticks and whole carcass meat for retail sale over a 1-year period; and (b) the genetic relatedness of human and chicken isolates collected concurrently. Enumeration of Campylobacter was performed using a spiral plater combined with manual spread plating. Campylobacter isolates were identified by polymerase chain reaction and typed by multilocus sequence typing (MLST). C. jejuni was the dominant species among both whole carcasses (63.5%) and drumsticks samples (73.8%), followed by C. coli (27% and 23.1%, respectively). After sample weight adjustment, whole carcasses showed significantly higher Campylobacter counts than drumsticks, with a significant difference in the counts between the commercial suppliers in both types of retail meat. MLST revealed 28 different sequence types among the two types of meat. Using permutational multivariate analysis of variance, statistically significant differences in the population genetic structures were observed between different suppliers but were not observed between the two types of chicken retail meat. In conclusion, we found differences in Campylobacter viable counts, suggesting consumption of whole carcasses may determine an exposure to a higher number of Campylobacter bacteria than consumption of chicken drumsticks. The Campylobacter population genetic structure did not differ between the two types of chicken retail meat. Therefore, source attribution studies based on MLST are unlikely to be biased by the selection of these types of retail meat during sampling.

Biofilm-Forming Potential of Staphylococcus aureus Isolated from Clinical Mastitis Cases in New Zealand.

Biofilm formation is of growing concern in human and animal health. However, it is still unclear how biofilms are related to mastitis infections in dairy cattle. In this study, a comparison between two tests for biofilm formation and the association between biofilm and the presence of genes associated with biofilm formation were investigated for 92 Staphylococcus aureus isolates from clinical mastitis cases. Congo red agar (CRA) and microtitre test assay (MTA) in vitro phenotypic tests were used to evaluate biofilm formation. The presence of icaA, icaD, and bap genes associated with biofilm formation was confirmed using the polymerase chain reaction. Results show that most of the S. aureus isolates, though not possessing one of the biofilm-forming genes, were able to produce biofilms. MTA was more frequently positive in identifying biofilm-forming isolates than CRA.