Amy V. Jennison

Identification of an unusual Brucella strain (BO2) from a lung biopsy in a 52 year-old patient with chronic destructive pneumonia

BackgroundBrucellosis is primarily a zoonotic disease caused by Brucella species. There are currently ten Brucella spp. including the recently identified novel B. inopinata sp. isolated from a wound associated with a breast implant infection. In this study we report on the identification of an unusual Brucella-like strain (BO2) isolated from a lung biopsy in a 52-year-old patient in Australia with a clinical history of chronic destructive pneumonia.ResultsStandard biochemical profiles confirmed that the unusual strain was a member of the Brucella genus and the full-length 16S rRNA gene sequence was 100% identical to the recently identified B. inopinata sp. nov. (type strain BO1T). Additional sequence analysis of the recA, omp2a and 2b genes; and multiple locus sequence analysis (MLSA) demonstrated that strain BO2 exhibited significant similarity to the B. inopinata sp. compared to any of the other Brucella or Ochrobactrum species. Genotyping based on multiple-locus variable-number tandem repeat analysis (MLVA) established that the BO2 and BO1Tstrains form a distinct phylogenetic cluster separate from the other Brucella spp.ConclusionBased on these molecular and microbiological characterizations, we propose that the BO2 strain is a novel lineage of the newly described B. inopinata species.

Vibrio cholerae O1 in 2 Coastal Villages, Papua New Guinea

To the Editor: Cholera outbreak reports are of international public health interest, especially in areas that were previously cholera free (1). Although many recent cholera outbreaks have originated in coastal areas (2), identifying the source of cholera introduction has been challenging (1). The detection of Vibrio cholerae in coastal, brackish and riverine waters in cholera-endemic and cholera-free areas supports the view that autochtonous V. cholerae is involved in the introduction of cholera (3,4). To our knowledge, cholera has not been reported in Papua New Guinea, despite social and environmental conditions likely to facilitate transmission and the nations close proximity to cholera-endemic countries (5,6). On August 6, 2009, a physician who visited the coastal village of Lambutina reported an outbreak of acute watery diarrhea that was associated with the death of his father and 4 other persons from this and a neighboring village. The outbreak began in the village of Nambariwa and spread to neighboring Lambutina, Morobe Province. From August 13, multidisciplinary teams worked with the community to reduce the number of deaths through early identification and treatment of case-patients. The teams also worked to limit transmission through improvements to the water and sanitation infrastructure and by encouraging better hygiene practices among the villagers. A suspected case of cholera was defined as acute watery diarrhea or vomiting in a resident of Lambutina or Nambariwa villages since July 22, 2009. In the 2 villages, 77 cases were identified; attack rates were 14% in Lambutina (48/343) and 5.5% in Nambariwa (29/532). The overall case-fatality ratio was 6.5% (5/77); 2 patients died after they were discharged from the referral hospital. A retrospective frequency-matched case–control study was conducted in Lambutina to identify the risk factors associated with suspected cholera. Neighborhood controls (± 5 years of age) were selected from unaffected households. Univariate and multivariate analyses were conducted with STATA version 10 (StataCorp., College Station, TX, USA). Of the 48 case-patients in Lambutina, 43 participated in the study with 43 age-matched controls. In addition to having close contact with patients who had cholera, univariate analysis showed that case-patients were more likely to have had several exposures related to the death of other patients (Table). However, having close contact with a patient was the only independent risk factor (adjusted odds ratio 4.8, 95% confidence interval 1.7–13.4) (Table). Close contact included providing nursing care for patients or carrying patients onto boats for transport to health care facilities. From the 10 collected samples, 4 isolates were confirmed as V. cholerae O1, biotype El Tor, serotype Ogawa, by PCR detection of an O1-specific region of the rfb gene using established methods and PCR amplification of the tcpA gene polymorphism specific for the El Tor biotype (7). The ctxAB, vct genes (present in toxigenic strains) and the hemolysin gene hlyA (present in all V. cholerae strains) were detected by PCR in all 4 isolates. Although health authorities promptly identified and responded to the outbreak, they could not determine its origin. The El Nino weather phenomenon generates increased rainfall and elevated sea surface temperatures and is a predictor of cholera outbreaks (8), which puts more coastal areas at risk for such outbreaks (9). During this outbreak, Papua New Guinea reported above-average rainfall (10) and warmer sea surface temperatures. Although cholera may have been introduced to Papua New Guinea through an infectious traveler or by other means, climatic factors may have initiated plankton blooms, the abundance of which have also been associated with increased presence of V. cholerae O1. Sea and estuarine waters of these villages are plausible sources of introduction. In Lambutina, the age-specific attack rates were lowest among young children and increased among persons of middle age and among the elderly. Those providing patient care and lifting during transportation as well as those washing the bodies of the deceased may have been more represented in the >40 years age group; however, this situation may not explain the high attack rates among the elderly. Generally, after a cholera outbreak is detected, interventions aim to reduce the proportion of deaths to <1%. The overall case-fatality ratio in the outbreak discussed here was 6.5%, which reflects the challenges to accessing adequate health care in remote settings. This difficulty is exacerbated when the disease occurs for the first time because cholera awareness and preparedness will be weak, as can be seen in the early management of cases during this outbreak. Villagers who have close contact with cholera patients are at greater risk for disease and should be a focus of interventions to limit transmission (e.g., eliminating ingestion of contaminated water, improving hygiene and sanitation). Education to increase awareness of the disease and enhanced access to low-osmolarity oral rehydration solution, Hartmann solution, and zinc supplements are essential. Cholera-endemic and cholera–nonendemic countries with coastal populations are at an increasing risk for cholera outbreaks. Adequate preparation by the health care system is vital to avoid excess deaths. Table Univariate and multivariate analysis of risk factors associated with suspected cholera in Lambutina village, Papua New Guinea, 2009*

Geographically Distinct Escherichia coli O157 Isolates Differ by Lineage, Shiga Toxin Genotype, and Total Shiga Toxin Production

ABSTRACT While the differential association of Escherichia coli O157 genotypes with animal and human hosts has recently been well documented, little is known about their distribution between countries and how this might affect regional disease rates. Here, we used a 48-plex single nucleotide polymorphism (SNP) assay to segregate 148 E. coli O157 isolates from Australia, Argentina, and the United States into 11 SNP lineages. We also investigated the relationship between SNP lineages, Shiga toxin (Stx) gene profiles, and total Stx production. E. coli O157 isolates clearly segregated into SNP lineages that were differentially associated with each country. Of the 11 SNP lineages, seven were detected among isolates from a single country, two were detected among isolates from all three countries, and another two were detected only among U.S. and Argentinean isolates. A number of Australian (30%) and Argentinean (14%) isolates were associated with novel, previously undescribed SNP lineages that were unique to each country. Isolates within SNP lineages that were strongly associated with the carriage of stx 2a produced comparatively more Stx on average than did those lacking the stx 2a subtype. Furthermore, the proportion of isolates in stx 2a-associated SNP lineages was significantly higher in Argentina and the United States than Australia (P < 0.05). This study provides evidence for the geographic divergence of E. coli O157 and for a prominent role of stx 2a in total Stx production. These results also highlight the need for more comprehensive studies of the global distribution of E. coli O157 lineages and the impacts of regionally predominant E. coli O157 lineages on the prevalence and severity of disease.

Extended-spectrum β-lactamase producing Escherichia coli in hospital wastewaters and sewage treatment plants in Queensland, Australia

We investigated the prevalence of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in untreated hospital wastewaters and 2 sewage treatment plants (STPs). A collection of 252 ESBL-producing E. coli isolates from hospital wastewater and STPs were typed and tested for resistance to 17 antimicrobial agents and for the presence of integron-associated integrases (intI gene) and ESBL genes. Eighty-nine percent (n = 176) of the ESBL-producing E. coli strains from hospital wastewater were found in more than 1 sample (common types), with 1 common type accounting for 35% of isolates, found in all samples. These strains were also resistant to up to 9 non-β-lactam antibiotics and showed the same pattern of resistance in all samples. More than 73% of the hospital wastewater isolates possessed SHV-type ESBL as opposed to isolates from STPs that carried only CTX-M-type ESBL genes. The prevalence of the intI gene did not differ between the sources of the isolates. Certain ESBL-producing E. coli were dominant in hospital wastewaters. These strains possessed β-lactamase genes that were different from isolates found in STPs. From a public health point of view, the presence of such a high level of ESBL-producing E. coli strains in hospital wastewaters is of great importance.

Cooperative Recognition of Internationally Disseminated Ceftriaxone-Resistant Neisseriagonorrhoeae Strain

Ceftriaxone remains a first-line treatment for patients infected by Neisseria gonorrhoeae in most settings. We investigated the possible spread of a ceftriaxone-resistant FC428 N. gonorrhoeae clone in Japan after recent isolation of similar strains in Denmark (GK124) and Canada (47707). We report 2 instances of the FC428 clone in Australia in heterosexual men traveling from Asia. Our bioinformatic analyses included core single-nucleotide variation phylogeny and in silico molecular typing; phylogenetic analysis showed close genetic relatedness among all 5 isolates. Results showed multilocus sequence type 1903; N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) 233; and harboring of mosaic penA allele encoding alterations A311V and T483S (penA-60.001), associated with ceftriaxone resistance. Our results provide further evidence of international transmission of ceftriaxone-resistant N. gonorrhoeae. We recommend increasing awareness of international spread of this drug-resistant strain, strengthening surveillance to include identifying treatment failures and contacts, and strengthening international sharing of data.

A Neisseria gonorrhoeae strain with a meningococcal mtrR sequence.

Genetic exchange between species within the Neisseria genus is well recognized (Linz et al., 2000) and can have important implications for the management of gonorrhoea, both in terms of detecting gonorrhoea by nucleic acid amplification tests and through the development of gonococcal antimicrobial resistance. For example, cross-reaction of commercial and in-house Neisseria gonorrhoeae nucleic acid amplification tests with commensal Neisseria species has been well documented (Farrell, 1999; Linz et al., 2000; Tabrizi et al., 2011). Furthermore, it is becoming increasingly clear that the acquisition of resistance determinants from commensal Neisseria species is one of the prime pathways leading to N. gonorrhoeae antimicrobial resistance; such determinants include the well-documented N. gonorrhoeae ‘mosaic’ penA sequences, which are thought to have been acquired from commensal Neisseria and are implicated in N. gonorrhoeae resistance to extended-spectrum cephalosporins (Ameyama et al., 2002; Unemo et al., 2012). Here, we report a novel ‘Neisseria meningitidis-like’ mtrR sequence in N. gonorrhoeae isolates with reduced susceptibility to azithromycin.

Molecular epidemiological typing of enteropathogenic Escherichia coli strains from Australian patients

Enteropathogenic Escherichia coli (EPEC) are an important cause of diarrhoea worldwide, particularly in children. Sixty-one EPEC strains isolated from stool specimens of symptomatic persons from 2008 to 2011 were characterised for the prevalence of diarrhoea-associated putative virulence genes. Phylogenetic typing, serotyping, multilocus variable-number repeat analysis (MLVA), and multilocus sequence typing (MLST) were also performed. The EPEC isolates were highly heterogeneous, representing all 4 phylogenetic groups and comprising 59 MLVA types, 49 MLST types, and 43 serotypes. This diversity is indicative of the complexity of the human enteric EPEC population, which may be either commensal or pathogenic.

Evaluation of the Meridian Premier EHEC assay as an indicator of Shiga toxin presence in direct faecal specimens

Molecular testing for stx1 and/or stx2 is a reliable way of detecting Shiga toxigenic Escherichia coli (STEC) when faecal specimens can be cultured; however, detection of Shiga toxin in unculturable specimens is also of public health importance. The Meridian Premier EHEC assay was evaluated against the gold standard Vero cell cytotoxic assay for Shiga toxin detection in direct faecal specimens. An initial study of 817 patient specimens submitted for routine STEC detection was conducted, evaluating positive faeces detected by the Meridian assay with the Vero cell assay. Twenty-nine percent of 136 Meridian-positive faeces were confirmed as containing Shiga toxin. A further 62 faecal specimens were evaluated for statistical purposes, with all specimens tested by both Meridian and Vero cell assays. On direct faeces, the Meridian assay gave high specificity (76.95%) but low sensitivity (40%). This study confirmed that testing by Meridian assay on cultures is preferential to testing direct faeces for Shiga toxin.

Prolonged and mixed non-O157 Escherichia coli infection in an Australian household

An Australian family was identified through a Public Health follow up on a Shiga-toxigenic Escherichia coli (STEC) positive bloody diarrhoea case, with three of the four family members experiencing either symptomatic or asymptomatic STEC shedding. Bacterial isolates were submitted to stx sequence sub-typing, multi-locus variable number tandem repeat analysis (MLVA), multi-locus sequence typing (MLST) and binary typing. The analysis revealed that there were multiple strains of STEC being shed by the family members, with similar virulence gene profiles and the same serogroup but differing in their MLVA and MLST profiles. This study illustrates the potentially complicated nature of non-O157 STEC infections and the importance of molecular epidemiology in understanding disease clusters.

Molecular characterization of an Australian serotype 1 Streptococcus pneumoniae outbreak

Serotype 1 Streptococcus pneumoniae is a cause of invasive pneumococcal disease (IPD) worldwide and has been associated with IPD outbreaks, while carriage is rarely detected in healthy adults or children. This study details an Australian multi-state and territory outbreak of serotype 1 S. pneumoniae IPD between 2010 and 2012. Molecular characterization demonstrated the outbreak was largely due to the clonal expansion of sequence type 306, MLVA type 261 S. pneumoniae serotype 1.