Increase in isolation of extended spectrum beta lactamase producing multidrug resistant non typhoidal Salmonellae in Pakistan
BMC Infectious Diseases
IRnesecarrceh aartsicele in isolation of extended spectrum beta lactamase producing multidrug resistant non typhoidal Salmonellae in Pakistan
Kauser Jabeen 0 1
Afia Zafar 0 1
Seema Irfan 0 1
Erum Khan 0 1
Vikram Mehraj 0 1
Rumina Hasan 0 1
0 This study was conducted from 1990-2006 at the Aga Khan University (AKU), a tertiary care hospital in Kara- chi, Pakistan. The hospital and laboratory are accredited with Joint commission of international accreditation (JCIA). Laboratory routinely participates in external
1 Department of Pathology and Microbiology, Aga Khan University , Karachi , Pakistan
Background: Increasing resistance to quinolones and ceftriaxone in non typhoidal Salmonellae is a global concern. Resistance to quinolone and 3rd generation cephalosporin amongst non typhoidal Salmonellae (NTS) from Pakistan has been reported in this study. Methods: Retrospective analysis of laboratory data was conducted (1990-2006). NTS were isolated and identified from clinical samples using standard microbiological techniques. Antimicrobial susceptibility testing was performed by Kirby Bauer. Extended spectrum beta lactamase production (ESBL) was detected using combined disc method. Ciprofloxacin sensitivity was detected by nalidixic acid screening method. Minimum inhibitory concentration (MIC) of ciprofloxacin was determined by agar dilution method. Statistical analysis was performed using SPSS version 13. Results: Analysis of 1967 NTS isolates showed a significant increase in ciprofloxacin resistance from 23% in 2002 to 50.5% in 2006, with increased mean MIC values from 0.6 to 1.3 ug/mL. Ceftriaxone resistant NTS also increased and ESBL production was seen in 98.7% isolates. These isolates exhibited high resistance against amoxicillin clavulanic acid (57%), gentamicin (69%), amikacin (44%) and piperacillin tazobactam (30%). No resistance to carbapenem was seen. Ceftriaxone resistance was significantly higher in children <1 year, in invasive isolates and in Salmonella Typhimurium. Conclusions: Increase in quinolone and ceftriaxone NTS is a serious threat to public health requiring continuous surveillance and use of appropriate screening tests for laboratory detection.
Non typhoidal salmonellae (NTS) are associated in
approximately 5% of the patients with bacteremia,
invasive infections and death . Increased antimicrobial
resistance has made empiric antibiotic of choice for these
potentially fatal infections quite limited. Current
literature recommends either a fluoroquinolones or a third
generation cephalosporins as drug of choice; however
increasing evidence of emergence of resistance against
these antibiotics in a great concern [2,3].
Outbreaks due to highly resistant NTS have been
reported from a number of countries [3-6]. Surveillance
of antimicrobial resistance amongst NTS is crucial,
particularly in developing countries where facilities for
culture and susceptibility testing are not widely accessible
and clinicians rely mainly on empirical therapy. While
increased resistance in typhoidal Salmonellae has been
reported from Pakistan, antimicrobial resistance data of
NTS is unavailable. Resistance to quinolone and 3rd
generation cephalosporin with extended spectrum beta
lactamase (ESBL) producing NTS have not been yet
reported from this region. We have assessed
antimicrobial susceptibility of NTS from Pakistan over a period of
17 years including analysis of resistance to quinolones
and 3rd generation cephalosporin.
quality control surveys with College of American
pathologists (CAP). Clinical microbiology laboratory receives
specimens across the country via satellite centers in 50
major cities and towns of Pakistan. The data presented
here was not collected in a programmed survey but
obtained in routine analysis of specimens submitted to
the laboratory. All specimens although were processed in
laboratory based in Karachi but the data represents
strains prevalent across the country. This study was a
retrospective analysis of the laboratory data and did not
include any identifiable information from the patients
therefore formal ethical committee approval was not
obtained as per research guidelines of the institute.
Data including both invasive and non-invasive NTS was
retrieved from a central computerized database.
Duplicate specimens from same patients and samples with
Salmonella enterica Typhi and Paratyphi were also excluded.
During the study period NTS from stool and urine were
identified biochemically using conventional tests .
Isolates from blood, tissues and other sterile body sites were
identified using API 20E (Bio Merieux France). Suspected
colonies were tested with Salmonella polyvalent antisera
(A-I and Vi) (Difco). Serogroups were determined using
the slide agglutination method. Non typeable Salmonella
spp. were those isolates that have biochemical (API 20E)
and serological profile suggestive of Salmonella but were
not further classified into serogroups due to limited
availability of specific O antisera (only A, B, C and D).
Serotypes were determined by tube agglutination method
against H antigen. If an isolate was not serotyped with the
available H antisera only serogroups were reported.
Antimicrobial susceptibility testing was performed as
per Clinical Laboratory Standards Institute (CLSI)
criteria  against nalidixic acid (30 g) ciprofloxacin (5 g),
ampicillin (10 g) ceftriaxone (30 g), chloramphenicol
(30 g), and cotrimoxazole (1.25/23.75 g). ESBL
production was detected by combined disc method (ceftriaxone
alone and ceftriaxone-clavulanic acid) . ESBL
producing strains were further tested against
amoxicillin-clavulanic acid (20/10 g), gentamicin (10 g), amikacin (30
g), piperacillin-tazobactam (100/10 g) and meropenem
(10 g). Since year 2002 susceptibility against
ciprofloxacin was determined by nalidixic acid screening method as
recommended by Hakanen et al . Minimum inhibitory
concentrations against ciprofloxacin were determined
using agar dilution method . Isolates that were
resistant to ampicillin, chloramphenicol and cotrimoxazole
were considered multidrug resistant (MDR).
Clinical data were analyzed using SPSS version 15.0
software. Comparisons were made between resistant and
sensitive strains in terms of age, gender, specimen source
and serogroups. Proportions were compared using the
Chi-square test or Fisher's exact test, where appropriate.
A p value of less than 5% was considered as statistically
During the study period, a total of 1967 NTS were
identified. A characteristic seasonal pattern in isolation of NTS
was observed with increased isolation in the months of
May-August corresponding with the summer and
monsoon season (data not shown). Majority of NTS isolates
were from females (57.5%). Isolation rate of NTS was
higher in the age group under 5 years (55.5%). Out of
1967 isolates majority were from stool (91%) followed by
blood (6%). Most prevalent organisms were the
Salmonella group B (659) including Salmonella enterica
Typhimurium (257) followed by Salmonella group C (465),
Salmonella group A (62) and Salmonella group D (48).
Due to limited availability of a number of antisera the
large group of non-typeable Salmonella (728) could not
The resistance rates against nalidixic acid, ofloxacin
and ceftriaxone were increased (Fig. 1). This increase was
significant for nalidixic acid and ofloxacin (p value for
trend < 0.001). Between 1990 and 2001 in accordance
with NCCLS guidelines interpretative criteria of
Enterobacteriaceae was used to report susceptibility of NTS
against fluoroquinolone, hence resistance was not
detected. However, following the introduction of
nalidixic acid screening method in 2002, 27% of the NTS
isolates were noted to have reduced susceptibility to
quinolones. Mean MIC values of ofloxacin in NTS also
gradually increased during this period (Fig 2).
Ceftriaxone resistant NTS first observed in year 2000,
increased gradually over the years. ESBL production was
seen in 98.7% (n = 78) of ceftriaxone resistant NTS. ESBL
producing NTS isolates further showed resistance against
amoxicillin clavulanic acid (57%), gentamicin (69%),
amikacin (44%) and piperacillin/tazobactam (30%).
Resistance to carbapenem was not observed. Ceftriaxone
resistance was significantly higher (p value < 0.001)
amongst organisms isolated from patients less than 1 year
of age, in invasive isolates, in hospitalized patients and in
Salmonella enterica Typhimurium. In comparison to
other Salmonella spp., Salmonella enterica Typhimurium
had significantly higher resistance to ciprofloxacin (35.5
vs. 19%) and had higher MDR rate (39% vs. 8%).
Interestingly resistance against the first line drugs did
not increase over the years. Rather resistance to
Figure 1 Showing marked increase in resistance against nalidixic acid, ofloxacin and ceftriaxone in NTS isolates at the Aga Khan University
phenicol decreased from 26% in 1990 to 7% in 2006 (p
value < 0.001). Similarly resistance against cotrimoxazole
and ampicillin remained static with minimum variations.
Isolation of MDR NTS strains significantly decreased to
3% in 2006 (p value < 0.001) (Fig 3).
Globally empirical therapy of invasive NTS infections has
become challenging with the emergence of ceftriaxone
and quinolone resistance. This is the first report from
Pakistan reporting ESBL producing NTS isolates and
increase in quinolone. We also observed recent decline in
isolation of MDR NTS strains.
Since 1991 ceftriaxone resistant NTS has been reported
in various studies [6,10] and is reportedly mainly due to
ESBL (particularly CTX-M types) and AmpC
lactamases [3,11]. Within our samples, ceftriaxone resistant NTS
were first isolated in the year 2000 and the rate of
isolation of these strains has gradually increased over the
years. 98.7% of our strains were ESBL producers and
showed concurrent resistance to broad spectrum
antibiotics including amoxicillin-clavulanic acid, amikacin,
gentamicin and piperacillin-tazobactam. Fortunately
carbapenem resistance, although reported previously in
NTS was not detected in our study . The significant
association of these strains with patients under than 1
year of age and with invasive infections makes the
management of these strains more problematic. Another
concern is inappropriate detection of such resistant strains in
most of the laboratories in Pakistan.
This study also reports an increase in fluoroquinolone
resistance from 23% in 2002 to 50.5% in 2006. Such an
increase is in agreement with reports from other
countries [4,5]. Prior to 2002 we were using CLSI breakpoints
Figure 2 Showing gradual increase in ciprofloxacin MICs in NTS isolates at the Aga Khan University Hospital (2002-2006).
recommended at the time and were therefore not able to
detect any resistance . From 2002 onwards nalidixic
acid screening method was employed for the detection of
quinolone resistance. The rise in resistance rates
correlates also with increase in mean MIC values for these
strains. We recommend that accurate detection of
Salmonella strains with reduced quinolone susceptibility should
be reported by the clinical laboratories in Pakistan, as
clinical outcome with quinolones therapy is poor.
Salmonella Typhimurium had highest resistance rates
to ceftriaxone, ciprofloxacin and MDR. Increased
resistance to first line drugs has been reported previously in
Salmonella Typhimurium from UK and Spain [13,14].
Another study from England and Wales has also reported
higher ceftriaxone resistance rates in this serotype .
Increased antimicrobial resistance in Salmonella
Typhimurium is alarming as high mortality and invasive
infections are associated with this serotype [15,16].
We also observed static resistance rates to ampicillin
and cotrimoxazole and a decline in resistance to
chloramphenicol and MDR strains as reported previously in
Kenya . Another report from Pakistan reporting
resistance rates in Salmonella Typhi also demonstrated
declining resistance to first line drugs . They
correlated this with the overall antimicrobial consumption at
population level in Karachi and showed a steady
reduction in the use of the above antibiotics especially
chloramphenicol. Based on their findings we can assume
similar correlation in NTS as well.
Increased resistance to ciprofloxacin and emergence of
ESBL producing NTS pose both diagnostic and
management dilemma in a developing country like Pakistan.
Measures should be taken to stop further emergence of
resistance and their dissemination on a priority basis.
Preventive strategy would require improved laboratory
services, continuous surveillance and restriction of use of
broad spectrum antibiotics.
The authors declare that they have no competing interests.
KJ initiated, planned and completed the study. She also wrote the initial
manuscript and SI, AZ, EK and RH critically analyzed it for intellectual content and
revised it accordingly. VM performed the data analysis. All authors approved
the final version.
Co tri mo x az o le
Chlo ram pheni col
Figure 3 Showing trends of resistance against first line drugs in NTS isolates at the Aga Khan University Hospital (1990-2006).
We would also like to thank the faculty and staff of the Clinical Microbiology
Laboratory Aga Khan University Hospital, Karachi for their support and help in
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