Prevalence and antibiotic susceptibility patterns of enteric bacterial pathogens in human and non-human sources in an urban informal settlement in Cape Town, South Africa.

BACKGROUND: In light of rampant childhood diarrhoea, this study investigated bacterial pathogens from human and non-human sources in an urban informal settlement. Meat from informal abattoirs (n = 85), river water (n = 64), and diarrheic stool (n = 66) were collected between September 2015 and May 2016. A duplex real-time PCR, gel-based PCR, and CHROMagar™STEC were used to screen Tryptic Soy Broth (TSB) for diarrheic E. coli. Standard methods were used to screen for other selected food and waterborne bacterial pathogens. RESULTS: Pathogens isolated from stool, meat, and surface water included Salmonella enterica (6, 5, 0%), Plesiomonas shigelloides (9, 0, 17%), Aeromonas sobria (3, 3, 0%), Campylobacter jejuni (5, 5, 0%), Shigella flexneri (17, 5, 0%), Vibrio vulnificus (0, 0, 9%), and diarrheic E. coli (21, 3, 7%) respectively. All the isolates were resistant to trimethoprim-sulphamethoxazole. CONCLUSIONS: There was a high burden of drug resistant diarrheal pathogens in the stool, surface water and meat from informal slaughter. Integrated control measures are needed to ensure food safety and to prevent the spread of drug resistant pathogens in similar settings.

Longitudinal Population Dynamics of Staphylococcus aureus in the Nasopharynx During the First Year of Life.

Background: Staphylococcus aureus colonization is a risk factor for invasive disease. Few studies have used strain genotype data to study S. aureus acquisition and carriage patterns. We investigated S. aureus nasopharyngeal carriage in infants in an intensively sampled South African birth cohort. Methods: Nasopharyngeal swabs were collected at birth and fortnightly from 137 infants through their first year of life. S. aureus was characterized by spa-typing. The incidence of S. aureus acquisition, and median carriage duration for each genotype was determined. S. aureus carriage patterns were defined by combining the carrier index (proportion of samples testing positive for S. aureus) with genotype diversity measures. Persistent or prolonged carriage were defined by a carrier index ≥0.8 or ≥0.5, respectively. Risk factors for time to acquisition of S. aureus were determined. Results: Eighty eight percent (121/137) of infants acquired S. aureus at least once. The incidence of acquisition at the species and genotype level was 1.83 and 2.8 episodes per child-year, respectively. No children had persistent carriage (defined as carrier index of >0.8). At the species level 6% had prolonged carriage, while only 2% had prolonged carriage with the same genotype. Carrier index correlated with the absolute number of spa-CCs carried by each infant (r = 0.5; 95% CI 0.35-0.62). Time to first acquisition of S. aureus was shorter in children from households with ≥5 individuals (HR 1.06, 95% CI 1.07-1.43), with S. aureus carrier mothers (HR; 1.5, 95% CI 1.2-2.47), or with a positive tuberculin skin test during the first year of life (HR; 1.81, 95% CI 0.97-3.3). Conclusion: Using measures of genotype diversity, we showed that S. aureus NP carriage is highly dynamic in infants. Prolonged carriage with a single strain occurred rarely; persistent carriage was not observed. A correlation was observed between carrier index and genotype diversity.

Proteomic comparison of three clinical diarrhoeagenic drug-resistant Escherichia coli isolates grown on CHROMagar™STEC media.

Shiga-toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) are key diarrhoea-causing foodborne pathogens. We used proteomics to characterize the virulence and antimicrobial resistance protein profiles of three clinical pathogenic E. coli isolates (two EPEC [one resistant to ciprofloxacin] and one STEC) cultured on CHROMagar™STEC solid media after minimal laboratory passage. We identified 4767 unique peptides from 1630 protein group across all three clinical E. coli strains. Label-free proteomic analysis allowed the identification of virulence and drug resistance proteins that were unique to each of the clinical isolates compared in this study. The B subunit of Shiga toxin, ToxB, was uniquely detected in the STEC strain while several other virulence factors including SheA, OmpF, OmpC and OmpX were significantly more abundant in the STEC strain. The ciprofloxacin resistant EPEC isolate possessed reduced levels of key virulence proteins compared to the ciprofloxacin susceptible EPEC and STEC strains. Parallel reaction monitoring assays validated the presence of biologically relevant proteins across biologically-replicated cultures. Propagation of clinical isolates on a relevant solid medium followed by mass spectrometry analysis represents a convenient means to quantify virulence factors and drug resistance determinants that might otherwise be lost through extensive in vitro passage in enteropathogenic bacteria. SIGNIFICANCE: Through the use of quantitative proteomics, we have characterized the virulence and antimicrobial resistance attributes of three clinically isolated, pathogenic E. coli strains cultured on solid media. Our results provide new, quantitative data on the expressed proteomes of these tellurite-resistant, diarrhoeagenic E. coli strains and reveal a subset of antimicrobial resistance and virulence proteins that are differentially abundant between these clinical strains. Our quantitative proteomics-based approach should thus have applicability in microbiological diagnostic labs for the identification of pathogenic/drug resistant E. coli in the future.

Development of a real-time PCR assay and comparison to CHROMagarTM STEC to screen for Shiga toxin-producing Escherichia coli in stool, Cape Town, South Africa.

INTRODUCTION: Shiga toxin-producing Escherichia coli (STEC) is an emerging infectious pathogen which could lead to haemolytic uremic syndrome. Even though previous studies have compared the performance of CHROMagarTMSTEC to real-time polymerase chain reaction (PCR) in Europe, no study has been done to assess its performance on African isolates. OBJECTIVES: This project aimed to validate and test an in-house-developed duplex real-time PCR and use it as a reference standard to determine the performance of CHROMagarTMSTEC on African isolates from diarrhoeic stool samples. METHODS: This study evaluated STEC diagnostic technology on African isolates. An in-house-developed duplex real-time PCR assay for detection of stx1 and stx2 was validated and tested on diarrhoeic stool samples and then used as a reference standard to assess the performance of CHROMagarTMSTEC. Real-time PCR was used to screen for stx in tryptic soy broth and the suspected STEC isolates, while conventional PCR was used to detect the other virulence genes possessed by the isolates. RESULTS: The real-time PCR limit of detection was 5.3 target copies/µL of broth. The mean melting temperature on melt-curve analysis for detection of stx1 was 58.2 °C and for stx2 was 65.3 °C. Of 226 specimens screened, real-time PCR detected stx in 14 specimens (6.2%, 95% confidence interval = 3.43% - 10.18%). The sensitivity, specificity, negative predictive value and positive predictive value of the CHROMagarTMSTEC were 33.3%, 77.4%, 95.3% and 11.3%. CONCLUSIONS: The in-house developed real-time PCR assay is a sensitive and specific option for laboratory detection of STEC as compared to CHROMagarTMSTEC in this setting.

Comparison of a Real-Time Multiplex PCR and Sequetyping Assay for Pneumococcal Serotyping.

BACKGROUND: Pneumococcal serotype identification is essential to monitor pneumococcal vaccine effectiveness and serotype replacement. Serotyping by conventional serological methods are costly, labour-intensive, and require significant technical expertise. We compared two different molecular methods to serotype pneumococci isolated from the nasopharynx of South African infants participating in a birth cohort study, the Drakenstein Child Health Study, in an area with high 13-valent pneumococcal conjugate vaccine (PCV13) coverage. METHODS: A real-time multiplex PCR (rmPCR) assay detecting 21 different serotypes/-groups and a sequetyping assay, based on the sequence of the wzh gene within the pneumococcal capsular locus, were compared. Forty pneumococcal control isolates, with serotypes determined by the Quellung reaction, were tested. In addition, 135 pneumococcal isolates obtained from the nasopharynx of healthy children were tested by both serotyping assays and confirmed by Quellung testing. Discordant results were further investigated by whole genome sequencing of four isolates. RESULTS: Of the 40 control isolates tested, 25 had a serotype covered by the rmPCR assay. These were all correctly serotyped/-grouped. Sequetyping PCR failed in 7/40 (18%) isolates. For the remaining isolates, sequetyping assigned the correct serotype/-group to 29/33 (88%) control isolates. Of the 132/135 (98%) nasopharyngeal pneumococcal isolates that could be typed, 69/132 (52%) and 112/132 (85%) were assigned the correct serotype/-group by rmPCR and sequetyping respectively. The serotypes of 63/132 (48%) isolates were not included in the rmPCR panel. All except three isolates (serotype 25A and 38) were theoretically amplified and differentiated into the correct serotype/-group with some strains giving ambigous results (serotype 13/20, 17F/33C, and 11A/D/1818F). Of the pneumococcal serotypes detected in this study, 69/91 (76%) were not included in the current PCV13. The most frequently identified serotypes were 11A, 13, 15B/15C, 16F and 10A. CONCLUSION: The rmPCR assay performed well for the 21 serotypes/-groups included in the assay. However, in our study setting, a large proportion of serotypes were not detected by rmPCR. The sequetyping assay performed well, but did misassign specific serotypes. It may be useful for regions where vaccine serotypes are less common, however confirmatory testing is advisable.

Ciprofloxacin-resistant Salmonella enterica serovar Kentucky in Canada.

We report emergence of ciprofloxacin-resistant Salmonella enterica serovar Kentucky in Canada during 2003-2009. All isolates had similar macrorestriction patterns and were multilocus sequence type ST198, which has been observed in Europe and Africa. Ciprofloxacin-resistant S. enterica serovar Kentucky represents 66% of all ciprofloxacin-resistant nontyphoidal Salmonella sp. isolates observed in Canada since 2003.

Emergence of multidrug-resistant Salmonella enterica serotype 4,[5],12:i:- involving human cases in Canada: results from the Canadian Integrated Program on Antimicrobial Resistance Surveillance (CIPARS), 2003-10.

OBJECTIVES: Over the last decade, a marked increase in Salmonella enterica serotype 4,[5],12:i:- with a core resistance to ampicillin, streptomycin, sulphonamides and tetracycline (ASSuT) has been observed in Europe. This study describes the emergence and characterization of isolates of multidrug-resistant Salmonella 4,[5],12:i:- in Canada. METHODS: Human clinical isolates of Salmonella 4,[5],12:i:- were identified by provincial laboratories from 2003 to 2010. Serotyping and phage typing were performed by standardized methodologies. MIC values were determined using broth microdilution. PCR was used to determine the presence of resistance genes. Multilocus sequence typing was performed on a selected number of isolates. RESULTS: A total of 26 251 Salmonella were submitted as part of the Canadian Integrated Program on Antibiotic Resistance Surveillance (CIPARS). Of these, Salmonella 4,[5],12:i:- accounted for a total of 766 isolates (2.9%), and the number increased significantly from 42 (1.4%) in 2003 to 164 (4.8%) in 2010. The ASSuT+ phenotype was observed in 11.9% (n = 91) of Salmonella 4,[5],12:i:- isolates and increased from two isolates in 2003 to 35 isolates in 2010. Two sequence types (STs) were observed. ST34 was mainly associated with the ASSuT isolates (n = 24; 38%), which contained blaTEM, strA-strB, tet(B) and sul2. ST19 was more likely to be associated with the ACSSuT phenotype and contained blaTEM, floR, strA-strB, sul2 and tet(A) or blaPSE-1, floR, aadA2, sul1 and tet(G). CONCLUSIONS: The prevalence of Salmonella 4,[5],12:i:- has significantly increased from 2003 to 2010 and it is now the fifth most common serotype reported in Canada causing human disease. Similar antimicrobial resistance patterns, phage types and STs have been observed in Europe.