John Merlino

Genetic analysis of Pseudomonas aeruginosa isolates from the sputa of Australian adult cystic fibrosis patients.

ABSTRACT 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 χ2 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.

Rapid Detection of Non-Multidrug-Resistant and Multidrug-Resistant Methicillin-Resistant Staphylococcus aureus Using Cycling Probe Technology for the mecA gene

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

Dissemination of Multiple Drug Resistance Genes by Class 1 Integrons in Klebsiella pneumoniae Isolates from Four Countries: a Comparative Study

ABSTRACT 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.

Diverse Mobilized Class 1 Integrons Are Common in the Chromosomes of Pathogenic Pseudomonas aeruginosa Clinical Isolates

ABSTRACT 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.

Limited diversity in the gene pool allows prediction of third-generation cephalosporin and aminoglycoside resistance in Escherichia coli and Klebsiella pneumoniae

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.

Tn6060, a Transposon from a Genomic Island in a Pseudomonas aeruginosa Clinical Isolate That Includes Two Class 1 Integrons

ABSTRACT A 25,441-bp transposon was recovered from a Pseudomonas aeruginosa clinical isolate. While the transposition module was >99% identical to sequence of Tn1403, 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.

Isolation and plasmid characterization of carbapenemase (IMP-4) producing Salmonella enterica Typhimurium from cats

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.

Plasmids and Bacterial Strains Mediating Multidrug-Resistant Hospital-Acquired Infections Are Coresidents of the Hospital Environment

Hospital-acquired infections (HAIs) are a global problem. The widespread use of antibiotics continues to exacerbate the problem giving rise to antibiotic-resistant bacteria both in and outside a clinical context. The general hospital environment is an obvious important focus for the selection and spread of multiresistant bacteria and a potential direct source of HAIs. Despite this, there are few detailed studies that have investigated the relationship between strains mediating HAIs and strains coresident in the hospital. Here we isolated bacteria from patients with HAIs exhibiting resistance to β-lactam antibiotics over a 1-month period in 2011. Three of these isolates were examined in detail by molecular analysis and their multiresistance regions were compared to β-lactam resistant bacteria isolated from the immediate hospital environment over the same period. All sampled patients were in a 14-bed burns unit and the environmental sample sites included shower drains, sinks, trolleys, and door handles. It was found that identical strains carrying the same resistance regions were present in both patients and the hospital environment suggesting HAIs can arise from bacteria resident in the immediate surrounds. The three patient infections were not derived from a single source, since strains could be distinguished by the genotype and spatial location. While it seems unlikely that eradication of multiresistant bacteria from the hospital can be achieved, more effective hospital cleaning and a better hospital design may be able to reduce transmission.

Comparative Analysis of Multidrug-Resistant, Non-Multidrug-Resistant, and Archaic Methicillin-Resistant Staphylococcus aureus Isolates from Central Sydney, Australia

ABSTRACT 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.

Resistance to the biocidal activity of silver in burn wound dressings - is it a problem?

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.