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Introduction. In this comparative case study, we discuss clinically-relevant discrepancies of antimicrobial susceptibility testing (AST) interpretation for ceftriaxone against a non-typable, beta-lactamase negative, ampicillin-resistant (BLNAR) Haemophilus influenzaeisolated from a blood culture. Case report: A 74 year old man presented with a 3-day illness characterised by shortness of breath and dry cough, and was noted to be febrile and hypoxic on admission. A blood culture bottle flagged positive with Gram-negative coccobacilli, later identified as Haemophilus influenzae with the patient commenced on ceftriaxone. The isolate was beta-lactamase negative and antibiotic susceptibility testing (AST) using disc diffusion revealed the isolate resistant to ceftriaxone and ampicillin by EUCAST methodology, with the patient subsequently changed to amoxicillin/clavulanate. Further AST using the CLSI methodology in parallel demonstrated discrepant results between the two susceptibility methods. The patient recovered without complications. Conclusion. This discrepancy could lead to inconsistent reporting of susceptibilities between laboratories, and consequently antibiotic prescribing, especially for invasive isolates. As more laboratories adopt EUCAST methodologies for AST interpretation in Australia and globally, it is important for clinicians to consider the clinical implications of these methodological discrepancies.

In Australia, the overuse or inappropriate prescribing of antimicrobials in health (human and animal) and agriculture is a concern that has increased over the years. This has given bacteria, fungi, parasites, and some viruses through exposure more opportunity to develop resistance. A process of artificial and natural selection, which favors microorganisms in developing the strongest natural defenses increasing prevalence of antimicrobial resistance (AMR). Appropriate use of antimicrobials can be lifesaving to humans and animals, but inappropriate use needs to be closely monitored and acted on to promote improved safety and quality of care. Inappropriate use includes prescribing antimicrobials when they are not necessary, prescribing the wrong type of antimicrobial and prescribing them for the incorrect duration. This short report reviews and summarizes some of the efforts used in Australia to control AMR and antibiotic prescribing.

The H30Rx subclade of Escherichia coli ST131 is a clinically important, globally dispersed pathogenic lineage that typically displays resistance to fluoroquinolones and extended spectrum β-lactams. Isolates EC233 and EC234, variants of ST131-H30Rx with a novel sequence type (ST) 8196, isolated from unrelated patients presenting with bacteraemia at a Sydney Hospital in 2014 are characterised here. EC233 and EC234 are phylogroup B2, serotype O25:H4A, and resistant to ampicillin, amoxicillin, cefoxitin, ceftazidime, ceftriaxone, ciprofloxacin, norfloxacin and gentamicin and are likely clonal. Both harbour an IncFII_2 plasmid (pSPRC_Ec234-FII) that carries most of the resistance genes on an IS26 associated translocatable unit, two small plasmids and a novel IncI1 plasmid (pSPRC_Ec234-I). SNP-based phylogenetic analysis of the core genome of representatives within the ST131 clonal complex places both isolates in a subclade with three clinical Australian ST131-H30Rx clade-C isolates. A MrBayes phylogeny analysis of EC233 and EC234 indicates ST8196 share a most recent common ancestor with ST131-H30Rx strain EC70 isolated from the same hospital in 2013. Our study identified genomic hallmarks that define the ST131-H30Rx subclade in the ST8196 isolates and highlights a need for unbiased genomic surveillance approaches to identify novel high-risk MDR E. coli pathogens that impact healthcare facilities.

The isolation of Neisseria zoodegmatis from a 63-year-old female presenting to the emergency department following a cat bite injury to her right hand is described in this report. N. zoodegmatis , also known as Centers for Disease Control (CDC) group EF-4b, is considered to be a zoonotic pathogen, and is usually associated with dog or cat bites. Despite the potential of this organism to cause serious soft tissue infections, it can be overlooked in routine clinical laboratories due to its slow growth characteristics and when the history of animal bite is not provided to the laboratory. This case highlights the importance of appropriate clinical history provision to the microbiology laboratory to help provide important information about potential pathogens and allow microbiologists to optimize culture and identification methods. The introduction of tools such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) into clinical laboratories allows identification and the interpretation of results to be performed within a few minutes of isolation on proper culture media, as opposed to traditional methods, whose slowness may be problematic, as shown in this case report.

Fosfomycin is a broad-spectrum antibiotic that targets UDP-Nacetylglucosamine enolpyruvyl transferase (MurA), an important enzyme in the early stages of peptidoglycan biosynthesis. In Escherichia coli, fosfomycin resistance can occur through the presence of fos genes (typically fosA3) which encode fosfomycininactivating enzymes (FosA, FosC2), or by mutations in proteins important for the uptake of fosfomycin (CyaA, GlpT, PtsI, UhpA, UhpT) or for its action (MurA)

This study sought to assess the genetic variability of Escherichia coli isolated from bloodstream infections (BSIs) presenting at Concord Hospital, Sydney during 2013-2016. Whole-genome sequencing was used to characterize 81 E. coli isolates sourced from community-onset (CO) and hospital-onset (HO) BSIs. The cohort comprised 64 CO and 17 HO isolates, including 35 multidrug-resistant (MDR) isolates exhibiting phenotypic resistance to three or more antibiotic classes. Phylogenetic analysis identified two major ancestral clades. One was genetically diverse with 25 isolates distributed in 16 different sequence types (STs) representing phylogroups A, B1, B2, C and F, while the other comprised phylogroup B2 isolates in subclades representing the ST131, ST73 and ST95 lineages. Forty-seven isolates contained a class 1 integron, of which 14 carried bla CTX -M-gene. Isolates with a class 1 integron carried more antibiotic resistance genes than isolates without an integron and, in most instances, resistance genes were localized within complex resistance loci (CRL). Resistance to fluoroquinolones could be attributed to point mutations in chromosomal parC and gyrB genes and, in addition, two isolates carried a plasmid-associated qnrB4 gene. Co-resistance to fluoroquinolone and broad-spectrum beta-lactam antibiotics was associated with ST131 (HO and CO), ST38 (HO), ST393 (CO), ST2003 (CO) and ST8196 (CO and HO), a novel ST identified in this study. Notably, 10/81 (12.3 %) isolates with ST95 (5 isolates), ST131 (2 isolates), ST88 (2 isolates) and a ST540 likely carry IncFII-IncFIB plasmid replicons with a full spectrum of virulence genes consistent with the carriage of ColV-like plasmids. Our data indicate that IncF plasmids play an important role in shaping virulence and resistance gene carriage in BSI E. coli in Australia.

Of 1033 Escherichia coli urinary tract infection isolates collected from females >12 years of age in Australia in 2019, only 2 isolates were resistant to fosfomycin with a minimum inhibitory concentration (MIC) of >256 mg/L. Despite having different multilocus sequence types, the two isolates harboured an identical plasmid-encoded fosA4 gene. The fosA4 gene has previously been identified in a single clinical E. coli isolate cultured in Japan in 2014. Each fosfomycin-resistant isolate harboured two conjugative plasmids that possessed an array of genes conferring resistance to aminoglycosides, β-lactams, macrolides, quinolones, sulfonamides and/or trimethoprim.

Objectives Implementation of EUCAST susceptibility testing in an Australian hospital laboratory demonstrated higher rates of aminopenicillin and amoxicillin/clavulanate resistance in Haemophilus influenzae than previously recognized. This study aimed to better define the variability in the detection of β-lactam resistance based on EUCAST and CLSI disc diffusion (DD) methodology, by comparison with the recommended reference method, broth microdilution (BMD), and by concordance with genomic analysis. Methods A total of 100 random H. influenzae isolates were assessed for ampicillin and amoxicillin/clavulanate susceptibility by EUCAST and CLSI DD and BMD. WGS was used to analyse the ftsI gene of a subset of isolates with β-lactam resistance, other than that due to isolated β-lactamase production. Results Of the 100 isolates, 32 were categorized as either β-lactamase negative, ampicillin resistant (BLNAR) (n = 18) or β-lactamase positive, amoxicillin/clavulanate resistant (BLPACR) (n = 14) by EUCAST DD. All 18 EUCAST BLNAR isolates were genotypically confirmed by WGS. Five of 18 BLNAR isolates were concordant by CLSI DD, 12 by EUCAST BMD and 4 by CLSI BMD. Nine of 14 EUCAST BLPACR isolates were confirmed by WGS; the remaining 5 were 1 mm below the EUCAST DD breakpoint. Only one isolate was detected as BLPACR by CLSI DD. Group III mutations associated with high-level ampicillin resistance were identified in 10/32 isolates. Conclusions The EUCAST DD susceptibility method is more reliable than either CLSI or BMD for the detection of genotypically defined BLNAR resistance. However, accurate categorization of amoxicillin/clavulanate resistance remains problematic. Continuous and reproducible surveillance of resistance is needed; for this to be possible, robust susceptibility methods are required.

IncHI2-ST1 plasmids play an important role in co-mobilizing genes conferring resistance to critically important antibiotics and heavy metals. Here we present the identification and analysis of IncHI2-ST1 plasmid pSPRC-Echo1, isolated from an Enterobacter hormaechei strain from a Sydney hospital, which predates other multi-drug resistant IncHI2-ST1 plasmids reported from Australia. Our time-resolved phylogeny analysis indicates pSPRC-Echo1 represents a new lineage of IncHI2-ST1 plasmids and show how their diversification relates to the era of antibiotics.

Carbapenem-resistant Enterobacteriaceae (CRE) are a pressing public health issue due to limited therapeutic options to treat such infections. CREs have been predominantly isolated from humans and environmental samples and they are rarely reported among companion animals. In this study we report on the isolation and plasmid characterization of carbapenemase (IMP-4) producing Salmonella enterica Typhimurium from a companion animal. Carbapenemase-producing S. enterica Typhimurium carrying blaIMP-4 was identified from a systemically unwell (index) cat and three additional cats at an animal shelter. All isolates were identical and belonged to ST19. Genome sequencing revealed the acquisition of a multidrug-resistant IncHI2 plasmid (pIMP4-SEM1) that encoded resistance to nine antimicrobial classes including carbapenems and carried the blaIMP-4-qacG-aacA4-catB3 cassette array. The plasmid also encoded resistance to arsenic (MIC-150 mM). Comparative analysis revealed that the plasmid pIMP4-SEM1 showed greatest similarity to two blaIMP-8 carrying IncHI2 plasmids from Enterobacter spp. isolated from humans in China. This is the first report of CRE carrying a blaIMP-4 gene causing a clinical infection in a companion animal, with presumed nosocomial spread. This study illustrates the broader community risk entailed in escalating CRE transmission within a zoonotic species such as Salmonella, and in a cycle that encompasses humans, animals and the environment.

New-onset postoperative atrial fibrillation (POAF) occurs in up to 30% of patients undergoing coronary artery bypass grafting (CABG). POAF is associated with short- and long-term mortality.To identify the true incidence and time course of recurrent atrial fibrillation (AF) in patients with POAF, we prospectively assigned 23 patients with POAF to receive an implantable loop recorder (ILR; Medtronic Inc., Minneapolis, MN, USA) for the detection of recurrent AF. Two electrophysiologists independently adjudicated monthly ILR transmissions to classify recurrent AF. We defined AF as any episode lasting ≥6 minutes.The cohort included 23 subjects averaging 69.1 ± 7.2 years of age. Their mean CHADSPOAF may represent a propensity for recurrent paroxysmal atrial fibrillation, and not simply a transient consequence of postoperative stress and inflammation. Better detection of recurrent AF might identify patients at risk for stroke who would benefit from continuing anticoagulation.

In this study, the phenotypic and genotypic characteristics of 50 methicillin-resistant Staphylococcus aureus (MRSA) isolates (43 contemporary and 7 archaic strains from the mid-1960s) from four Sydney hospitals in the central Sydney area were compared. Phenotypic analysis based on antibiotic profiles and phage typing patterns categorized the MRSA isolates into three major groups: multidrug resistant (mMRSA), non-multidrug resistant (nmMRSA), and archaic. The nmMRSA isolates could be further subdivided into nmMRSA group 1, which was phage typeable and similar to the archaic group; nmMRSA group 2, which was non-phage typeable and only resistant to ciprofloxacin; and nmMRSA group 3, which was also nontypeable and generally resistant to other antibiotics. The characterization of all five phenotypic groups was then extended by genetic analysis. Restriction fragment length polymorphism (RFLP) analysis showed the 50 isolates could be sorted into 20 group-specific pulsotypes. mecI gene deletions and mutations at various percentages among the five MRSA groups were detected by sequencing. Several mec promoter mutations were also found. The overall findings indicated that nmMRSA strains may have independently acquired mec DNA and are more likely to be newly emergent strains than nmMRSA variants.

Ninety clinical Staphylococcus aureus isolates from separate patients were examined phenotypically and genotypically for susceptibility to methicillin/oxacillin. Thirty were methicillin/oxacillin susceptible and 60 were methicillin and oxacillin resistant (MRSA). The 60 MRSA isolates examined were subdivided into two groups according to their antibiotic profiles and comprised 30 non-multidrug-resistant (NMDR) isolates, resistant to less than two non-beta-lactam antibiotics, and 30 multidrug-resistant (MDR) isolates, resistant to three or more non-beta-lactam antibiotics. Phenotypic and genotypic analysis of methicillin/oxacillin showed that despite use of the guidelines published by the NCCLS for the testing of S. aureus susceptibility to methicillin/oxacillin, MIC values of some NMDR MRSA isolates fell below the NCCLS-recommended breakpoints. Etest strips failed to detect two NMDR MRSA isolates tested with oxacillin and four tested with methicillin. Lowering the NCCLS-recommended oxacillin screen agar concentration from 6 to 2 mg/L and temperature of incubation to 30 degrees C, improved the specificity and sensitivity of NMDR MRSA detection from 87% to 100%. On PFGE analysis these NMDR MRSA strains were genotypically different. Genotypic tests, such as multiplex PCR for the mecA/nuc genes and DNA hybridization for the mecA gene, or phenotypic monoclonal antibody-based tests to detect penicillin-binding protein 2a (PBP2a) offer advantages for problematic isolates in detecting or confirming low-level phenotypic heterogeneous mecA expression of oxacillin and methicillin resistance in NMDR MRSA.

Antimicrobial resistance is a complex issue and is a threat to public health globally. While emerging and re-emerging diseases have captured news headlines with outbreaks such SARS, bird flu, Ebola and Zika virus around the world, leading health experts tells us that there is a more serious threat to public health – antimicrobial resistance (AMR)1.

Plasmid-mediated class C β-lactamases are reported from Escherichia coli and Klebsiella pneumoniae with increasing frequency. No screening and confirmatory tests have been uniformly established for these strains. We investigated for the presence of plasmid-mediated AmpC production in 51 clinical isolates of Enterobacteriaceae, comparing two different boronic acid formulations, phenylboronic acid (PB) and 3-(N-Boc-amino)phenylboronic acid (APB), using polymerase chain reaction (PCR). PB performed better than APB.

Early appropriate antibiotic treatment reduces mortality in severe sepsis, but current methods to identify antibiotic resistance still generally rely on bacterial culture. Modern diagnostics promise rapid gene detection, but the apparent diversity of relevant resistance genes in Enterobacteriaceae is a problem. Local surveys and analysis of publicly available data sets suggested that the resistance gene pool is dominated by a relatively small subset of genes, with a very high positive predictive value for phenotype. In this study, 152 Escherichia coli and 115 Klebsiella pneumoniae consecutive isolates with a cefotaxime, ceftriaxone and/or ceftazidime minimum inhibitory concentration (MIC) of ≥ 2 μg/mL were collected from seven major hospitals in Sydney (Australia) in 2008-2009. Nearly all of those with a MIC in excess of European Committee on Antimicrobial Susceptibility Testing (EUCAST) resistance breakpoints contained one or more representatives of only seven gene types capable of explaining this phenotype, and this included 96% of those with a MIC ≥ 2 μg/mL to any one of these drugs. Similarly, 97% of associated gentamicin-non-susceptibility (MIC ≥ 8 μg/mL) could be explained by three gene types. In a country like Australia, with a background prevalence of resistance to third-generation cephalosporins of 5-10%, this equates to a negative predictive value of >99.5% for non-susceptibility and is therefore suitable for diagnostic application. This is an important proof-of-principle that should be tested in other geographic locations.

Antimicrobial resistance is a major problem for hospitals and the community. This interactive session developed for trainees will explore current antimicrobial resistance issues pertinent to the laboratory, including detection and importance in determining mechanisms of resistance, screening, therapeutic implications and consequences for infection control. Four trainees will present illustrative cases of antimicrobial resistance (MRSA, VRE, multi-resistant Gram-negative organisms and Candida ) followed by expert panel discussion. Dr John Merlino will deliver an update on hot topics in antimicrobial susceptibility testing and resistance in a ‘show and tell session'. Finally trainees and the expert panel will have an opportunity to discuss prepared questions on antimicrobial resistance issues.

Eleven clinical class 1 integron-containing Pseudomonas aeruginosa isolates from Australia and Uruguay were investigated for the genomic locations of these elements. Several novel class 1 integrons/transposons were found in at least four distinct locations in the chromosome, including genomic islands. These elements seem to be undergoing successful dispersal by lateral gene transfer since integrons were identified across several lineages and more than one clonal line.

A comparative genetic analysis of 42 clinical Klebsiella pneumoniae isolates, resistant to two or more antibiotics belonging to the broad-spectrum β-lactam group, sourced from Sydney, Australia, and three South American countries is presented. The study focuses on the genetic contexts of class 1 integrons, mobilizable genetic elements best known for their role in the rapid evolution of antibiotic resistance among Gram-negative pathogens. It was found that the class 1 integrons in this cohort were located in a number of different genetic contexts with clear regional differences. In Sydney, IS26-associated Tn21-like transposons on IncL/M plasmids contribute greatly to the dispersal of integron-associated multiple-drug-resistant (MDR) loci. In contrast, in the South American countries, Tn1696-like transposons on an IncA/C plasmid(s) appeared to be disseminating a characteristic MDR region. A range of mobile genetic elements is clearly being recruited by clinically important mobile class 1 integrons, and these elements appear to be becoming more common with time. This in turn is driving the evolution of complex and laterally mobile MDR units and may further complicate antibiotic therapy. Fil: Chowdhury, Piklu Roy. University of Technology; Australia Fil: Ingold, Ana. Universidad de la República; Uruguay Fil: Vanegas, Natasha. University of Technology; Australia. Universidad El Bosque; Colombia Fil: Martínez, Elena. University of Technology; Australia. Universidad de la República; Uruguay Fil: Merlino, John. Concord Hospital; Australia. University of Sydney; Australia Fil: Merkier, Andrea Karina. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: Castro, Mercedes. Servicio de Sanidad de las Fuerzas Armadas; Uruguay Fil: Rocha, Gerardo González. Universidad de Concepción; Chile Fil: Borthagaray, Graciela. Universidad de la República; Uruguay Fil: Centron, Daniela. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina Fil: Toledo, Helia Bello. Universidad de Concepción; Chile Fil: Márquez, Carolina M.. Universidad de la República; Uruguay Fil: Stokes, H. W.. University of Technology; Australia

In clinical laboratories the phenotypic detection of low-level vancomycin resistance in enterococci is problematic. Rapid detection of vancomycin resistant enterococci (VRE), in particular those that carry the vanA or vanB genes, may minimize transmission within a hospital and therefore reduce the risk of colonization and infection.

A 25,441-bp transposon was recovered from a Pseudomonas aeruginosa clinical isolate. While the transposition module was >99% identical to sequence of Tn 1403 , the element had been subject to rearrangements, with two In70.2-like class 1 integrons inserted into it in an unusual “tail-to-tail” configuration. One cassette array was the same as that in In70.2; however, the second was different, generating a transposon that collectively includes six resistance cassettes.

The ASM Antimicrobial Special Interest Group (ASIG) is made up of colleagues and ASM members you have an interest in antimicrobials. Members of the ASIG committee include medical, veterinary, CDS Users, mycology and parasitology consultants. The Antimicrobial SIG is one of the largest Special Interest Groups in the ASM. In 2012 we had over 923 ASIG members. In 2013 we had 721 members listed under ASIG. Some of our members have retired. Numbers are yet to be finalised in 2014.

This report describes a new, modified, simple, and cost-effective method for the use of CHROMagar Candida (CHROMagar Company, Paris, France) for the presumptive identification of isolates as Candida albicans after preliminary growth. Sixty randomly selected clinical isolates were evaluated, including 38 of C. albicans . With incubation at 37°C for 24 h, the sensitivity and specificity appeared to be excellent and the test performed better than the traditional germ tube test. However, at earlier times, C. tropicalis isolates gave false-positive results.

Genetic investigations were carried out with 50 phenotypically selected strains of Pseudomonas aeruginosa from 18 patients attending an Australian cystic fibrosis (CF) center. The isolates were analyzed by restriction fragment length polymorphism (RFLP) analysis by pulsed-field gel electrophoresis (PFGE). Phylogenetic analysis of the macrorestriction patterns showed rates of genetic similarity ranging from 76 to 100%; 24 (48%) of the strains from 11 patients had greater than 90% similarity. A dominant strain emerged: 15 isolates from seven patients had identical PFGE patterns, and 4 other isolates were very closely related. The 50 isolates were grouped into 21 pulsotypes on the basis of visual delineation of a three-band difference. Ten of the 18 (56%) patients were infected with clonal or subclonal strains. Sequence analysis of PCR products derived from the mucA gene showed 20 mutations, with the number of mutations in individual isolates ranging from 1 to 4; 19 of these changes are reported here for the first time. Potentially functional changes were found in 22 (44%) isolates. Eight changes (five transversions and three single base deletions) led to premature stop codons, providing support for the presence of mucA mutations as one pathway to mucoidy. There was a trend toward an association between the dominant strain and lack of potentially functional mucA mutations ( P = 0.09 by the χ 2 test) but no relationship between genotype and phenotype. This is the first study of genetic variation in P. aeruginosa isolates from adult Australian CF patients. The findings highlight the need for further investigations on the transmissibility of P. aeruginosa in CF patients.

TheAntimicrobial Special InterestGroup (ASIG) isoneof the largest special interest groupswithin the ASM. In 2012 there were over 923 ASIG members. In 2013 there were over 721 members listed under ASIG. Some of our members have retired but still keep in touch. In 2012–13 ASM ASIG members were actively involved with local and national ASM Branch meetings in various states, usually in the form of communication and newsletters. The committee actively communicates via emails and the website www.asig.org.au on emerging issues, methodology on susceptibility testing, standards on antimicrobials and antimicrobial resistance.

Lyn Gilbert , Jon Iredell and John Merlino Marie Bashir Institute for Emerging Infections and Biosecurity, Centre for Values, Ethics and the Law in Medicine, University of Sydney, and Level 3, Institute of Clinical Pathology & Medical Research, Westmead Hospital, PO Box 533, Wentworthville, NSW 2145, Australia. Tel: +61 2 9845 6252, Email: lyn.gilbert@sydney.edu.au WestmeadMillennium Institute forMedical Research, C24 –WestmeadHospital, TheUniversity of Sydney, NSW2006, Australia. Tel: +61 2 9845 6255, Email: jonathan.iredell@sydney.edu.au Department of Microbiology and Infectious Diseases, Concord Hospital, and Department of Infectious Diseases and Immunology, Sydney Medical School, University of Sydney, Hospital Road, Concord, NSW 2139, Australia. Tel: +61 2 9767 6658, Email: JMerlino@med.usyd.edu.au

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This paper describes a new chromogenic plate medium, CHROMagar Staph aureus (CHROMagar, Paris, France), for the identification of Staphylococcus aureus on the basis of colony pigmentation. The abilities of CHROMagar Staph aureus, thermostable nuclease (DNase), and mannitol salt agar (MSA) to identify S. aureus isolates ( n = 114) and discriminate between S. aureus and coagulase-negative staphylococci (CoNS; n = 22) were compared. CHROMagar Staph aureus proved to be more sensitive and specific than DNase and MSA, allowing a reliable, simple, and rapid method for the identification of S. aureus isolates. All CoNS encountered in this study with the exception of S. chromogenes could be easily differentiated from S. aureus on this medium. The supplementation with 4 μg of oxacillin or methicillin per ml allowed simple identification of methicillin resistance in hospital-acquired S. aureus strains which show multiple-drug resistance profiles. Community-acquired methicillin-resistant S. aureus strains showing non-multi-drug resistance profiles require further evaluation on this new chromogenic medium. Methicillin or oxacillin resistance of all S. aureus isolates was confirmed by the detection of penicillin-binding protein 2a, encoded by the mecA gene, using the latex slide agglutination MRSA-Screen test (PBP 2′ Test, DR900M; Oxoid).

Lipovitellin-salt-mannitol (LSM) plate medium was examined for its ability to directly isolate, recover, and presumptively identify Staphylococcus aureus from 418 clinical specimens. The criteria for medium evaluation included colony morphology reactions, selectivity, and ease of isolation. For 298 specimens used for screening, LSM agar medium was compared with the other conventional media used, mannitol salt agar (MSA), 5% horse blood agar (HBA), and phenolphthalein phosphate agar (PPA), to detect and recover S. aureus and methicillin-resistant S. aureus. The results indicated that LSM agar is more effective than MSA, HBA, or PPA for the recovery and isolation of S. aureus and methicillin-resistant S. aureus. On a replicator multipoint inoculation system, we compared the reactions on LSM agar, MSA, and DNase agar of 227 different strains of staphylococci, which included 178 different strains of S. aureus and 49 different strains of coagulase-negative staphylococci isolated from clinical specimens. By using the lipovitellin precipitation activity and mannitol fermentation characteristics, LSM agar gave a 100% correlation in presumptively identifying S. aureus. LSM agar may be an alternative plate medium for large hospital extensive screening for the detection and isolation of S. aureus.

A new chromogenic plate medium, CHROMagar Orientation, was evaluated for use in the differentiation and presumptive identification of gram-negative bacilli and Enterococcus species by a multipoint inoculation (replicator) technique. In this study, 1,404 gram-negative bacilli and 74 enterococcal isolates were tested on CHROMagar Orientation. Six control American Type Culture Collection strains were also included with the testing to ensure quality control of the media. Of the Escherichia coli isolates (n = 588) tested, 99.3% produced a pink-to-red color. Only in four isolates that were O-nitrophenyl-beta-D-galactopyranoside (ONPG) negative did this result differ. Proteus mirabilis and P. vulgaris were well differentiated on this medium. P. mirabilis (n = 184) produced a clear colony with diffusible brown pigment around the periphery. By contrast, 15 of 16 P. vulgaris isolates produced bluish-green colonies with a slight brown background. All Aeromonas hydrophila isolates (n = 26) tested produced clear to pink colonies at 35 to 37 degrees C. This colony color changed to blue after 2 to 3 h of incubation at room temperature. A. hydrophila exhibited stronger color and better growth at 30 degrees C. Serratia marcescens (n = 29) demonstrated an aqua blue color that deepened to a darker blue when exposed to room temperature. All enterococcal isolates (n = 74) resulted in a blue color and gave pinpoint colonies on purity subcultures at 35 to 37 degrees C after 18 h of incubation. Similarity in color resulted in failure to discriminate accurately between Klebsiella, Enterobacter, and Citrobacter species. However, these species could be readily differentiated from other members of the family Enterobacteriaceae. Pseudomonas aeruginosa (n = 151) was easily differentiated from members of the Enterobacteriaceae but was less easily distinguishable from other gram-negative nonmembers of the Enterobacteriaceae. The medium was found to facilitate easy visual detection of mixed bacterial isolates in culture. When used in a replicator system, it easily detected mixed growths of organisms which may have otherwise led to false antibiotic susceptibility results. These mixed growths were not obvious on the routine susceptibility testing medium (Isosensitest).

In clinical laboratories rapid and accurate detection and identification of methicillin/oxacillin resistance in Staphylococcus aureus (MRSA) is essential for the management and treatment of both colonised and infected community and hospitalised patients [1, 2, 3]. In this study, we evaluated a new DNA probe assay, the Velogene-Cycling Probe Technology (CPT) assay (ID Biomedical; Australian Laboratory Services Sydney, Australia), for its ability to detect rapidly methicillin or oxacillin resistance in Staphylococcus aureus isolates showing multidrug resistance (MDR) with homogeneous high-level resistance to methicillin/oxacillin and nonmultidrug resistance (NMDR) with heterogeneous lowlevel methicillin/oxacillin resistance. Infections with heterogeneous NMDR MRSA are sometimes difficult to detect phenotypically due to growth-dependent factors characteristic of certain strains and geographical diversity imposed by selective antibiotic pressures [2, 3]. A total of 90 clinical Staphylococcus aureus isolates were tested. The types of isolates and their minimum inhibitory concentration (MIC) values for oxacillin and methicillin were as follows: 30 methicillin-susceptible Staphylococcus aureus (MSSA) with oxacillin MICs of 0.19 �g/ml–1.5 �g/ml and methicillin MICs of 0.75 �g/ ml–1.5 �g/ml; 30 MDR MRSA with oxacillin and methicillin MIC values greater than or equal to 256 �g/ ml; and 30 NMDR MRSA with oxacillin MIC values of 1.5 �g/ml to greater than 256 �g/ml and methicillin MIC values of 4 �g/ml to greater than 256 �g/ml. Reference strains MSSA ATCC 25923 (mecA negative) and MRSA ATCC 43300 (mecA positive) were used as controls. MICs were determined using E test strips (AB Biodisk, Sweden) and broth dilutions as previously described [3]. All isolates were obtained from separate patients and checked for genetic diversity by pulsed-field gel electrophoresis/restriction fragment length polymorphism typing [3]. The multiplex polymerase chain reaction (PCR) for the mecA and nuc genes was the gold standard method used for MSSA and MRSA identification [3, 4, 5]. The Velogene-CPT assay was performed in accordance with the manufacturer’s instructions, except the Staphylococcus aureus colonies were taken from a 5% horse blood

The ability to rapidly detect and identify micro-organisms is of paramount importance for many microbiology laboratories. The application of new classes of enzymatic-based chromogenic compounds has revolutionised traditional culture media, leading to the developments of a new generation of chromogenic media. Many studies have shown that the chromogenic agar has become more than an isolation media when compared to conventional culture media. A newer, faster, more cost-effective means in the presumptive identification or screening of microorganisms which are either genus, or in some cases species-specific, and allows superior differentiation of mixed micro-organisms in cultures. When supplemented with antibiotics or other agents, chromogenic media allows the screening for multidrug resistance and virulence in many micro-organisms and can be easily integrated with other automated and/or molecular-based methods.

Biocides, whether naturally extracted or chemically engineered, are invaluable compounds that provide the health industry with numerous benefits. They can be used as disinfectants or antiseptics playing an important role in the control of bacteria and viruses. Thus, they are a precious resource that must be managed so as to be protected from loss of activity over time; these compounds are often overlooked in the significance of bacterial persistence and resistance.

Severe burn injuries are commonly associated with significant mortality and morbidity. A burn injury of 30% of the body surface area is associated with generalised depression of the immune system. Survival from these injuries is due to many factors, including the control of bacterial colonisation and infections leading to sepsis. Many of the organisms commonly recovered from infected patients in the burn ICU are members of the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) group of pathogens recognised as the most challenging bacteria carrying multidrug (MDR) resistance facing clinicians today. Efforts to control wound burn sepsis is routinely managed by the topical application of dressings containing silver. There is concern that some microorganisms can develop resistance to the biocidal activity of silver and that this may increase due to the widespread commercial use of silver.

Robert Koch quickly recognised that there was no universal environmental condition which would support the growth of all bacteria. Bacterial cells within clonal populations can show phenotypic variation due to internal stochastic processes. Variation between individual cells can be beneficial as well as detrimental to the survival of a population exposed to stress such as change in environmental conditions and many other variables such as selective antibiotic pressure. Variation in the level of proteins relevant for growth or survival among individual cells in a population is likely to play a role in drug resistance and disease. Cells reproduce by incorporating metabolite molecules and die at rates dependent on their gene expression and environmental states.